Foreign DNA detection by high-throughput sequencing to regulate genome-edited agricultural products.
ABSTRACT: Although the advent of several new breeding techniques (NBTs) is revolutionizing agricultural production processes, technical information necessary for their regulation is yet to be provided. Here, we show that high-throughput DNA sequencing is effective for the detection of unintended remaining foreign DNA segments in genome-edited rice. A simple k-mer detection method is presented and validated through a series of computer simulations and real data analyses. The data show that a short foreign DNA segment of 20 nucleotides can be detected and the probability that the segment is overlooked is 10-3 or less if the average sequencing depth is 30 or more, while the number of false hits is less than 1 on average. This method was applied to real sequencing data, and the presence and absence of an external DNA segment were successfully proven. Additionally, our in-depth analyses also identified some weaknesses in current DNA sequencing technologies. Hence, for a rigorous safety assessment, the combination of k-mer detection and another method, such as Southern blot assay, is recommended. The results presented in this study will lay the foundation for the regulation of NBT products, where foreign DNA is utilized during their generation.
Project description:OBJECTIVE:To quantitatively assess the factors associated with non-beneficial treatments (NBTs) in hospital admissions at the end of life. DESIGN:Retrospective multicentre cohort study. SETTING:Three large, metropolitan tertiary hospitals in Australia. PARTICIPANTS:831 adult patients who died as inpatients following admission to the study hospitals over a 6-month period in 2012. MAIN OUTCOME MEASURES:Odds ratios (ORs) of NBT derived from logistic regression models. RESULTS:Overall, 103 (12.4%) admissions involved NBTs. Admissions that involved conflict within a patient's family (OR 8.9, 95%?CI 4.1 to 18.9) or conflict within the medical team (OR 6.5, 95%?CI 2.4 to 17.8) had the strongest associations with NBTs in the all subsets regression model. A positive association was observed in older patients, with each 10-year increment in age increasing the likelihood of NBT by approximately 50% (OR 1.5, 95%?CI 1.2 to 1.9). There was also a statistically significant hospital effect. CONCLUSIONS:This paper presents the first statistical modelling results to assess the factors associated with NBT in hospital, beyond an intensive care setting. Our findings highlight potential areas for intervention to reduce the likelihood of NBTs.
Project description:Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status.Gene expression profiling of 49 diagnostic primary NBTs with ploidy data was performed using oligonucleotide microarray. Further analyses using Quantitative Real-Time Polymerase Chain Reaction (Q-PCR); array-Comparative Genomic Hybridization (aCGH); and Fluorescent in situ Hybridization (FISH) were performed to investigate the correlation between aneuploidy, chromosomal changes and gene expression profiles.Gene expression profiling of 49 primary near-triploid and near-diploid/tetraploid NBTs revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (P = 0.01) and 17p13-q21 (P < 0.0001), described as recurrently altered in NBTs. Over 90% of these genes showed higher expression in near-triploid NBTs and the majority are involved in cell differentiation pathways. Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. Comparison between gene copy number and expression levels suggests that differential expression might be only partly dependent on gene copy number. Intratumoural clonal heterogeneity was observed in all NBTs, with marked interclonal variability in near-diploid/tetraploid tumours.NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results demonstrate that these specific genetic abnormalities are highly heterogeneous in all NBTs, and suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis.
Project description:We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the nbtA and nbtE genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the nbtS gene revealed that this gene encoded a salicylate synthase. These results indicate that the nbt clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the nbtA, -G, -H, -S, and -T genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that nbt genes are regulated coordinately by IdeR protein in an iron-dependent manner. The ?nbtE mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of N. farcinica.
Project description:Neuroblastic tumours (NBTs) represent a heterogeneous spectrum of neoplastic diseases associated with multiple genetic alterations. Structural and numerical chromosomal changes are frequent and are predictive parameters of NBTs outcome. We performed a comparative analysis of the biological entities constituted by NBTs with different ploidy status. Gene expression profiling of 49 diagnostic primary near-triploid (n= 22) and near-diploid/tetraploid (n=27) NBTs performed using Affymetrix U95Av2 arrays, revealed distinct expression profiles associated with each NBT subgroup. A statistically significant portion of genes mapped to 1p36 (P=0.01) and 17p13-q21 (P<0.0001). Specific chromosomal abnormalities observed in NBTs, 1p loss, 17q and whole chromosome 17 gains, were reflected in the gene expression profiles. This study demonstrates that NBTs with different cellular DNA content display distinct transcriptional profiles with a significant portion of differentially expressed genes mapping to specific chromosomal regions known to be associated with outcome. Furthermore, our results suggest that NBTs with different ploidy status may result from different mechanisms of aneuploidy driving tumourigenesis. Overall design: lavar-00109 Assay Type: Gene Expression Provider: Affymetrix Array Designs: HG_U95Av2 Organism: Homo sapiens (ncbitax) Tissue Sites: Adrenal Gland Material Types: total_RNA Cell Types: primary tumor Disease States: Neuroblastoma
Project description:We successfully realised plasmon-driven selective reduction reactions of 2-amino-5-nitrobenzenethiol (2A-5-NBT) to 3,3'-dimercapto-4,4'-diaminoazobenzene , an azobenzene derivative, using surface-enhanced Raman scattering (SERS) spectroscopy, and supported by the theoretical calculations. The SERS spectra demonstrated that two 5-nitro groups of 2A-5-NBTs were selectively reduced to the -N=N- chemical bond of 3,3'-dimercapto-4,4'-diaminoazobenzene, whereas the 2-amine group of 2A-5-NBT remained unchanged. Our experimental results revealed that aqueous environments were preferable to ambient atmospheric environments for this selective reduction reaction. The product is very stable in aqueous environments. However, in ambient atmosphere environments, the product is not stable and can revert back to 2A-5-NBT, where the -N=N- chemical bond can be broken by plasmon scissors. The plasmon-induced catalytic reactions in aqueous environments could be used for the efficient synthesis of aromatic azobenzene derivative compounds, which are valuable chemicals that are widely used in the chemical industry as dyes, food additives and drugs.
Project description:The pod-shaped TiO2 nano burst tubes (TiO2 NBTs) were prepared by the combination of electrospinning and impregnation calcination with oxalic acid (H2C2O4), polystyrene (PS) and tetrabutyl titanate. The silver nanoparticles (AgNPs) were loaded onto the surface of TiO2 NBTs by ultraviolet light reduction method to prepare pod-shaped Ag@TiO2 NBTs. In this work, we analysed the effect of the amount of oxalic acid on the cracking degree of TiO2 NBTs; the effect of the concentration of AgNO3 solution on the particle size and loading of AgNPs on the surface of TiO2 NBTs. Scanning electron microscopy and transmission electron microscopy investigated the surface morphology of samples. X-ray diffraction and X-ray photoelectron spectroscopy characterized the structure and composition of samples. Rhodamine B (RhB) solution was used to evaluate the photocatalytic activity of pod-shaped TiO2 NBTs and Ag@TiO2 NBTs. The results showed that TiO2 NBTs degraded 91.0% of RhB under ultraviolet light, Ag@TiO2 NBTs degraded 95.5% under visible light for 75 and 60 min, respectively. The degradation process of both samples was consistent with the Langmuir-Hinshelwood first-order kinetic equation. Therefore, the catalytic performance of the sample is: Ag@TiO2 NBTs > TiO2 NBTs > TiO2 nanotubes.
Project description:Reconciling sustainability with agricultural productivity in the face of climate change relies strongly on the development of resilient, high-yielding crops of superior nutritional value that can be grown more resource efficiently. Therefore, innovation in plant breeding has gained unprecedented importance. Plant breeding depends upon genetic variability within crops and their relatives as a basis for developing new plant varieties with improved characteristics. Plant breeders are continuously integrating the latest methods in plant biology and genetics into their breeding toolbox to more efficiently use existing diversity but also to induce new genetic variation. Over the past years, ever more precise and efficient plant breeding methods have been developed. This plant breeding innovation leap is based on an in-depth understanding of plant genomes and refinement of breeding methods, enabling more efficient, more precise and faster progress in achieving the desired breeding goals. Consequently, these plant breeding innovations are rapidly being developed and utilized internationally and across the seed sector, public and private research, plant species and markets. The results of a survey among 62 private plant breeding companies conducted by Euroseeds and presented in this publication confirm the enormous interest of companies in using new breeding techniques (NBTs) for a wide range of crop species and traits and the negative impact of the current regulatory situation in the EU on companies' decisions for investments in NBT-related R&D activities for the EU market and beyond.
Project description:Innovation in agriculture is pervasive. However, in spite of the success stories of twentieth century plant breeding, the twenty-first century has ushered in a set of challenges that solutions from the past century are unlikely to address. However, sustained research and the amalgamation of a number of disciplines has resulted in new breeding techniques (NBTs), such as genome editing, which offer the promise of new opportunities to resolve some of the issues. Here we present the results of an expert survey on the added potential benefits of genome-edited crops compared to those developed through genetic modification (GM) and conventional breeding. Overall, survey results reveal a consensus among experts on the enhanced agronomic performance and product quality of genome-edited crops over alternatives. The majority of experts indicated that the regulations for health and safety, followed by export markets, consumers, and the media play a major role in determining where and how NBTs, including genome editing, will be developed and used in agriculture. Further research is needed to gauge expert opinion after the Court of Justice of the European Union ruling establishing that site-specific mutagenic breeding technologies are to be regulated in the same fashion as GM crops, regardless of whether foreign DNA is present in the final variety.
Project description:Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGS-based clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking. Bioinformatics analysis workflows based on k-mers have been shown to allow for fast and efficient analysis of large genomics data sets as obtained from microbial sequencing applications. We here demonstrate applicability of k-mer based clinical microbiology assays for whole-genome sequencing (WGS) including variant calling, taxonomic identification, bacterial typing as well as AMR marker detection. The wet-lab and dry-lab workflows were developed and validated in line with Clinical Laboratory Improvement Act (CLIA) guidelines for laboratory-developed tests (LDTs) on multi-drug resistant ESKAPE pathogens. The developed k-mer based workflow demonstrated ?99.39% repeatability, ?99.09% reproducibility and ?99.76% accuracy for variant calling and applied assays as determined by intra-day and inter-day triplicate measurements. The limit of detection (LOD) across assays was found to be at 20× sequencing depth and 15× for AMR marker detection. Thorough benchmarking of the k-mer based workflow revealed analytical performance criteria are comparable to state-of-the-art alignment based workflows across clinical microbiology assays. Diagnostic sensitivity and specificity for multilocus sequence typing (MLST) and phylogenetic analysis were 100% for both approaches. For AMR marker detection, sensitivity and specificity were 95.29 and 99.78% for the k-mer based workflow as compared to 95.17 and 99.77% for the alignment-based approach. Summarizing, results illustrate that k-mer based analysis workflows enable a broad range of clinical microbiology assays, potentially not only for WGS-based typing and AMR gene detection but also genetic prediction of antibiotic susceptibility testing results.
Project description:The remarkable plasticity of Schwann cells (SCs) is essential for nerve regeneration but also contributes to neuropathies and cancer progression. It has not yet been investigated whether the adaptive potential of SCs is manifested in stromal, tumor associated SCs characteristically found within a benign subtype of neuroblastic tumors (NBTs). We here performed transcriptome profiling of human NBTs, rich and poor in SC stroma, as well as human injured nerves, rich in repair SCs, revealing that stromal SCs exhibit a repair SC characteristic gene expression signature. In turn, primary repair SCs had a pro-differentiating and anti-proliferative effect on NBT cell lines after direct and trans-well co-culture. Within the pool of secreted stromal/repair SC factors, we identified EGFL8, a matricellular protein with so far undescribed function, to induce neuronal differentiation of aggressive NBT cells. EGFL8 expression further correlated with favorable tumor stage and increased patient survival. Our findings suggest that stromal SCs exert nerve repair associated functions in the tumor-environment and underline the therapeutic value of SC-derived factors for aggressive, SC stroma-poor NBTs.