From Benchtop to Desktop: Important Considerations when Designing Amplicon Sequencing Workflows

Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose

Sequencing of idiopathic pulmonary fibrosis-related genes reveals independent single gene associations

BACKGROUND: Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes. OBJECTIVE: To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server. METHODS: We resequenced the genes encoding surfactant proteins A2 (SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare (minor allele frequency <1%), computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF. RESULTS: We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant. CONCLUSIONS: Rare mutations in SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation

Initiating e-learning by stealth, participation and consultation in a late majority institution

The extent to which opportunities afforded by e-learning are embraced by an institution can depend in large measure on whether it is perceived as enabling and transformative or as a major and disruptive distraction. Most case studies focus on the former. This paper describes how e-learning was introduced into the latter environment. The sensitivity of competing pressures in a research intensive university substantially influenced the manner in which e-learning was promoted. This paper tells that story, from initial stealth to eventual university acknowledgement of the relevance of e-learning specifically to its own context

AffecTech- an affect-aware interactive AV Artwork

New developments in real-time computing and body-worn sensor technology allow us to explore not just visible gestures using inertial sensors, but also invisible changes in an individual’s physiological state using bio-sensors (Kim & André 2008). This creates an opportunity for a more intimate interaction between the observer and technology-based art (Gonsalves 2008). We present a technical overview of the AffecTech system; a bio-signal based interactive audio- visual installation commissioned as part of the pre-ISEA symposium in November 2008. Observers were invited to sit on one of 2 sensor-enhanced chairs (Coghlan & Knapp 2008), which transmitted physiological data about the occupant to a central control system. This data was used to control and modulate interactive visuals, live video feeds and a surround sound score, with events and interactions dependent on the observers’ affective/emotional state and the disparity or similarity between the bio-signals of the chairs occupants. This technical overview is followed by an examination of the outcomes of the project, from both the artistic and technical viewpoints, with recommendations for modification in future implementations

A unique 3D nitrogen-doped carbon composite as high-performance oxygen reduction catalyst

The synthesis and properties of an oxygen reduction catalyst based on a unique 3-dimensional (3D) nitrogen doped (N-doped) carbon composite are described. The composite material is synthesised via a two-step hydrothermal and pyrolysis method using bio-source low-cost materials of galactose and melamine. Firstly, the use of iron salts and galactose to hydrothermally produceiron oxide (Fe₂O₃) magnetic nanoparticle clusters embedded carbon spheres. Secondly, magnetic nanoparticles diffused out of the carbon sphere when pyrolysed in the presence of melamine as nitrogen precursor. Interestingly, many of these nanoparticles, as catalyst-grown carbon nanotubes (CNTs), resulted in the formation of N-doped CNTs and N-doped carbon spheres under the decomposition of carbon and a nitrogen environment. The composite material consists of integrated N-doped carbon microspheres and CNTs show high ORR activity through a predominantly four-electron pathway.Ramesh Karunagaran, Tran Thanh Tung, Cameron Shearer, Diana Tran, Campbell Coghlan, Christian Doonan, and Dusan Losi

Neonatal abstinence syndrome.

A 12 month review of infants admitted with neonatal abstinence syndrome to a neonatal intensive care unit was undertaken. The relationship of maternal drug abuse to symptoms, the effectiveness of pharmacologic agents in controlling symptoms and the length of inpatient stay were investigated. A retrospective review of maternal and infant records was performed. Those infants with a serial Finnegan score greater than 8 were treated. Pharmacologic treatment was oral morphine sulphate (0.2 mg 4-6 hourly), phenobarbitone (3-7 mgs/kg/day), or combination of the above. 43 infants were admitted to the hospital during the year. The average maternal age was 24.6 years, (18-34 years). Drug use volunteered by the mothers was methadone alone in 6 cases, methadone and benzodiazepines in 14, methadone and heroin and benzodiazepines in 7, methadone and heroin in 10, heroin alone in 2, and other multiple drug use including oral morphine sulphate, dothiepin and cannabis in 4. Average gestational age was 40.3 (35-42 weeks). The average birthweight was 2.81 kgs (1.89-3.91 kgs). Time to onset of withdrawal symptoms was 2.8 (1-13) days. The duration of pharmacologic treatment (oral morphine sulphate and/or phenobarbitone) was 21.8 (1-62) days. The total hospital stay for the 43 infants was 1,011 days. This study confirms that polydrug abuse is the commonest type of drug abuse in Dublin. The duration of withdrawal symptoms is loosely related to drug type, but increasing duration of symptoms is noted for infants exposed to benzodiazepines. Our experience would favour the use of morphine sulphate to treat pure opiate withdrawal symptoms. Over the 12-month period, there was an average occupancy of 3 beds per day in the paediatric department

The Effect of Physiological Cyclic Stretch on the Cell Morphology, Cell Orientation and Protein Expression of Endothelial Cells

In vivo, endothelial cells are constantly exposed to pulsatile shear and tensile stresses. The main aim of this study was to design and build a physiological simulator, which reproduced homogenous strain profiles of the tensile strain experienced in vivo, and to investigate the effect of this cyclic tensile strain on the cell morphology, cell orientation and protein expression of endothelial cells. The biological response of human umbilical vein endothelial cells to a uniaxial cyclic stretch, in this newly developed simulator, was examined experimentally using immunohistostaining and confocal imaging and it was found that the cells elongated and oriented at 58.9± 4.5. This value was compared to a mathematical model where it was revealed that endothelial cells would orient at an angle of 60. This model also revealed that endothelial cells have an axial strain threshold value of 1.8% when exposed to a 10% cyclic strain at 1 Hz for 3 h. Cells cultured under conditions of cyclic strain showed increased ICAM-1 immunostaining when compared to static cells whereas, a marked decrease in the levels of VCAM-1 receptor staining was also observed. Haemodynamic stresses can modulate the endothelial cell adhesion response in vivo thus, taken together; this data validates the bioreactor as replicating the physiological environment

Dilemmas in doing insider research in professional education

This article explores the dilemmas I encountered when researching social work education in England as an insider researcher who was simultaneously employed as an educator in the host institution. This was an ethnographic project deploying multiple methods and generating rich case study material which informed the student textbook Becoming a Social Worker the four-year period of the project. First, ethical dilemmas emerged around informed consent and confidentiality when conducting surveys of students and reading their portfolios. Second, professional dilemmas stemmed from the ways in which my roles as a researcher, academic tutor, social worker and former practice educator converged and collided. Third, political dilemmas pertained to the potential for the project to crystallize and convey conflicts among stakeholders in the university and community. Since the majority of research in social work education is conducted by insiders, we have a vital interest in making sense of such complexity

Architectural Education in Unfamiliar Territory: The Fish River Aboriginal Ranger Accommodation Project

The Fish River Aboriginal Ranger Accommodation project presents a case study of an experiential learning approach where design and construction are undertaken in unfamiliar environmental and cultural conditions to expand the learning experience beyond the traditional boundaries of architectural education. The project offers an example of an integrated pedagogy, entwining traditional design studio with technical, social and cultural experiences. The teaching and learning journey of this project required students and staff to expand their professional and personal skills beyond the drawings of the design studios and into the cultural and climatic context of a remote area of northern Australia.David Kroll, David Morris and Joti Weijers-Coghlan, Arianna Brambill

A City in Common: A Framework to Orchestrate Large-scale Citizen Engagement around Urban Issues

Citizen sensing is an approach that develops and uses lightweight technologies with local communities to collect, share and act upon data. In doing so it enables them to become more aware of how they can tackle local issues. We report here on the development and uptake of the 'City- Commons Framework for Citizen Sensing', a conceptual model that builds on Participatory Action Research with the aim of playing an integrating role: outlining the processes and mechanisms for ensuring sensing technologies are co-designed by citizens to address their concerns. At the heart of the framework is the idea of a city commons: a pool of community-managed resources. We discuss how the framework was used by communities in Bristol to measure and monitor the problem of damp housing