Fluorescent amplification for next generation sequencing (FA-NGS) library preparation.

BACKGROUND:Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process. RESULTS:In this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data. CONCLUSIONS:We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types

Linguistic incompetence: giving an account of researching multilingually

This paper considers the place of linguistic competence and incompetence in the context of researching multilingually. It offers a critique of the concept of competence and explores the performative dimensions of multilingual research and its narration, through the philosophy of Judith Butler, and in particular her study Giving an account of oneself. It explores aspects of risk, justice, narrative limit and a morality of multilingualism in emergent multilingual research frameworks. These theoretical dimensions are explored through consideration of ‘linguistically incompetent’ ethnographic work with refugees and asylum seekers, in contexts of hospitality and in life long learning research in the Gaza Strip, and of early attempts to learn new languages. The paper offers a prospect of a relational approach to researching multilingually and affirms the vulnerability at the heart of linguistic hospitality

Physical aspects of oracles for randomness, and Hadamard's conjecture

We analyze the physical aspects and origins of currently proposed oracles for (absolute) randomness.Comment: 10 pages, 3 figures. arXiv admin note: substantial text overlap with arXiv:1405.140

Evaluating the effects of bilingual traffic signs on driver performance and safety

Variable Message Signs (VMS) can provide immediate and relevant information to road users and bilingual VMS can provide great flexibility in countries where a significant proportion of the population speak an alternative language to the majority. The study reported here evaluates the effect of various bilingual VMS configurations on driver behaviour and safety. The aim of the study was to determine whether or not the visual distraction associated with bilingual VMS signs of different configurations (length, complexity) impacted on driving performance. A driving simulator was used to allow full control over the scenarios, road environment and sign configuration and both longitudinal and lateral driver performance was assessed. Drivers were able to read one and two-line monolingual signs and two-line bilingual signs without disruption to their driving behaviour. However, drivers significantly reduced their speed in order to read four-line monolingual and four-line bilingual signs, accompanied by an increase in headway to the vehicle in front. This implies that drivers are possibly reading the irrelevant text on the bilingual sign and various methods for reducing this effect are discussed

Prediction of extreme events in the OFC model on a small world network

We investigate the predictability of extreme events in a dissipative Olami-Feder-Christensen model on a small world topology. Due to the mechanism of self-organized criticality, it is impossible to predict the magnitude of the next event knowing previous ones, if the system has an infinite size. However, by exploiting the finite size effects, we show that probabilistic predictions of the occurrence of extreme events in the next time step are possible in a finite system. In particular, the finiteness of the system unavoidably leads to repulsive temporal correlations of extreme events. The predictability of those is higher for larger magnitudes and for larger complex network sizes. Finally, we show that our prediction analysis is also robust by remarkably reducing the accessible number of events used to construct the optimal predictor.Comment: 5 pages, 4 figure

Overall survival in the OlympiA phase III trial of adjuvant olaparib in patients with germline pathogenic variants in BRCA1/2 and high-risk, early breast cancer

Adjuvant therapy; Breast cancer; OlaparibTerapia adyuvante; Cáncer de mama; OlaparibTeràpia adjuvant; Càncer de mama; OlaparibBackground The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. Patients and methods One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. Results With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI −0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. Conclusion With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals.Funding for this work, which was conducted as a collaborative partnership among the Breast International Group, NRG Oncology, Frontier Science, AstraZeneca, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, U.S.A. (MSD), was provided by the National Institutes of Health (grant numbers: U10CA 180868, UG1CA 189867, and U10CA 180822) and by AstraZeneca as part of an alliance between AstraZeneca and MSD. Provision of olaparib and placebo was from AstraZeneca

Spectral engineering of optical fiber preforms through active nanoparticle doping

Europium doped alkaline earth fluoride [Eu:AEF(2) (AE = Ca, Sr, Ba)] nanoparticles were synthesized and systematically incorporated into the core of modified chemical vapor deposition (MCVD)-derived silica-based preforms by solution doping. The resulting preforms were examined to determine the impact of the nanoparticles chemistry on the spectroscopic behavior of the glass. The dominant existence of Eu3+ was demonstrated in all preforms, which is in contrast to conventional solution doped preforms employing dissolved europium salts where Eu2+ is primarily observed. Raman spectroscopy and fluorescence lifetime measurements indicated that the nanoparticles composition is effective in controlling, at a local chemical and structural level, the spectroscopic properties of active dopants in optical fiber glasses. Further, there is a systematic and marked increase in radiative lifetime, tau, of the Eu3+ emission that follows the cationic mass; tau(Ca) \u3c tau(Sr) \u3c tau(Ba) with the BaF2-derived sample yielding a 37% lengthening of the lifetime over the CaF2-derived one. Such nanoscale control of what otherwise is silica glass could be useful for realizing property-enhanced and tailored spectroscopic performance from otherwise standard materials, e.g., vapor-derived silica, in next generation optical fibers

Spectral Engineering of Optical Fiber Preforms Through Active Nanoparticle Doping

Europium doped alkaline earth fluoride [Eu:AEF2 (AE = Ca, Sr, Ba)] nanoparticles were synthesized and systematically incorporated into the core of modified chemical vapor deposition (MCVD)-derived silica-based preforms by solution doping. The resulting preforms were examined to determine the impact of the nanoparticles chemistry on the spectroscopic behavior of the glass. The dominant existence of Eu3+ was demonstrated in all preforms, which is in contrast to conventional solution doped preforms employing dissolved europium salts where Eu2+ is primarily observed. Raman spectroscopy and fluorescence lifetime measurements indicated that the nanoparticles composition is effective in controlling, at a local chemical and structural level, the spectroscopic properties of active dopants in optical fiber glasses. Further, there is a systematic and marked increase in radiative lifetime, τ, of the Eu3+ emission that follows the cationic mass; τCa \u3c τSr \u3c τBa with the BaF2-derived sample yielding a 37% lengthening of the lifetime over the CaF2-derived one. Such nanoscale control of what otherwise is silica glass could be useful for realizing property-enhanced and tailored spectroscopic performance from otherwise “standard” materials, e.g., vapor-derived silica, in next generation optical fibers