The application of genomic technologies to cancer and companion diagnostics.

This thesis describes work undertaken by the author between 1996 and 2014. Genomics is the study of the genome, although it is also often used as a catchall phrase and applied to the transcriptome (study of RNAs) and methylome (study of DNA methylation). As cancer is a disease of the genome the rapid advances in genomic technology, specifically microarrays and next generation sequencing, are creating a wave of change in our understanding of its molecular pathology. Molecular pathology and personalised medicine are being driven by discoveries in genomics, and genomics is being driven by the development of faster, better and cheaper genome sequencing. The next decade is likely to see significant changes in the way cancer is managed for individual cancer patients as next generation sequencing enters the clinic. In chapter 3 I discuss how ERBB2 amplification testing for breast cancer is currently dominated by immunohistochemistry (a single-gene test); and present the development, by the author, of a semi-quantitative PCR test for ERBB2 amplification. I also show that estimating ERBB2 amplification from microarray copy-number analysis of the genome is possible. In chapter 4 I present a review of microarray comparison studies, and outline the case for careful and considered comparison of technologies when selecting a platform for use in a research study. Similar, indeed more stringent, care needs to be applied when selecting a platform for use in a clinical test. In chapter 5 I present co-authored work on the development of amplicon and exome methods for the detection and quantitation of somatic mutations in circulating tumour DNA, and demonstrate the impact this can have in understanding tumour heterogeneity and evolution during treatment. I also demonstrate how next-generation sequencing technologies may allow multiple genetic abnormalities to be analysed in a single test, and in low cellularity tumours and/or heterogenous cancers. Keywords: Genome, exome, transcriptome, amplicon, next-generation sequencing, differential gene expression, RNA-seq, ChIP-seq, microarray, ERBB2, companion diagnostic

The accommodation of contested identities: The impact of participation in a practice-based Masters Programme on Beginning Teachers’ professional identity and sense of agency.

Abstract Teachers’ professional training and development has been the focus of intense academic and political debate. This paper contributes to this by considering Beginning Teachers’ (BTs’) self-views of their professional identity. The findings are derived from a mixed methods study with questionnaires (n=886) and focus groups and interviews (n= 60) with BTs in Wales. Drawing on a socio-cultural approach, the findings illustrate how BTs’ integration of competing professional identities bolstered their sense of professional agency. These findings have salience within a policy context where both teacher education and professional development are increasingly aligned with the narrow organizational objectives of the school. Keywords: teacher professionalism; sociology of professions; socio-cultural theory; Beginning Teachers; professional development; professional identity; teacher agency

Bioanalyzer chips can be used interchangeably for many analyses of DNA

The Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) enables small-scale gel electrophoretic separation of nucleic acids on a microfluidic chip. Shortage of chips and excess reagents is a common issue. This report explored the compatibility of two commonly used Bioanalyzer reagents with three Bioanalyzer chip types. Microfluidic electrophoretic separation of DNA and RNA using DNA High Sensitivity and RNA 6000 Nano reagents, respectively, was successfully performed on multiple chip types, following the assay-specific protocol. For RNA quality and next-generation sequencing library size estimation, the Bioanalyzer chip s tested can be used interchangeably. These findings will be valuable for any laboratory using the Agilent Bioanalyzer in a shared facility.We thank all members of the Genomics Core team past and present for useful discussions; Sarah Vowler for help with statistical tests; and Cancer Research UK and the University of Cambridge for funding the Genomics Core facilities through the Cambridge Institute grant

The cost of reducing starting RNA quantity for Illumina BeadArrays: a bead-level dilution experiment.

BACKGROUND: The demands of microarray expression technologies for quantities of RNA place a limit on the questions they can address. As a consequence, the RNA requirements have reduced over time as technologies have improved. In this paper we investigate the costs of reducing the starting quantity of RNA for the Illumina BeadArray platform. This we do via a dilution data set generated from two reference RNA sources that have become the standard for investigations into microarray and sequencing technologies. RESULTS: We find that the starting quantity of RNA has an effect on observed intensities despite the fact that the quantity of cRNA being hybridized remains constant. We see a loss of sensitivity when using lower quantities of RNA, but no great rise in the false positive rate. Even with 10 ng of starting RNA, the positive results are reliable although many differentially expressed genes are missed. We see that there is some scope for combining data from samples that have contributed differing quantities of RNA, but note also that sample sizes should increase to compensate for the loss of signal-to-noise when using low quantities of starting RNA. CONCLUSIONS: The BeadArray platform maintains a low false discovery rate even when small amounts of starting RNA are used. In contrast, the sensitivity of the platform drops off noticeably over the same range. Thus, those conducting experiments should not opt for low quantities of starting RNA without consideration of the costs of doing so. The implications for experimental design, and the integration of data from different starting quantities, are complex.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Developing the capacity to support beginning teachers in Wales: Lessons learnt from the Masters in Educational Practice

One of the key requirements for any system level reform to be effective is to ensure that ‘at the point of delivery’ the necessary capacity is available to those responsible for its implementation (Mourshed, 2010; Robinson et al., 2011). The Masters in Educational Practice (MEP) was a key element of the Welsh Government’s strategy for professional development and school improvement. The paper does not seek to evaluate the MEP programme’s effectiveness (forthcoming) but, instead, considers its design and ‘enactment’ (Ball, Maguire & Braun, 2012). The paper approached the issues of the enactment of the MEP: how the programme was shaped by the interaction of individual and collective agency in overlapping contexts, from the theoretical perspective of capacity building at a system level (Hadfield and Chapman, 2009). The analytical focus of the paper is on the nature of the temporary intermediate organisation (Asheim, 2002) constructed to lead the implementation of the MEP and the challenges it faced in accessing, cohering and aligning sufficient capacity from within, and external to, the Welsh education system

Mycoplasma genitalium: whole genome sequence analysis, recombination and population structure.

BACKGROUND: Although Mycoplasma genitalium is a common sexually transmitted pathogen causing clinically distinct diseases both in male and females, few genomes have been sequenced up to now, due mainly to its fastidious nature and slow growth. Hence, we lack a robust phylogenetic framework to provide insights into the population structure of the species. Currently our understanding of the nature and diversity of M. genitalium relies on molecular tests targeting specific genes or regions of the genome and knowledge is limited by a general under-testing internationally. This is set against a background of drug resistance whereby M. genitalium has developed resistance to mainly all therapeutic antimicrobials. RESULTS: We sequenced 28 genomes of Mycoplasma genitalium from temporally (1980-2010) and geographically (Europe, Japan, Australia) diverse sources. All the strain showed essentially the same genomic content without any accessory regions found. However, we identified extensive recombination across their genomes with a total of 25 regions showing heightened levels of SNP density. These regions include the MgPar loci, associated with host interactions, as well as other genes that could also be involved in this role. Using these data, we generated a robust phylogeny which shows that there are two main clades with differing degrees of genomic variability. SNPs found in region V of 23S rRNA and parC were consistent with azithromycin/erythromycin and fluoroquinolone resistances, respectively, and with their phenotypic MIC data. CONCLUSIONS: The sequence data here generated is essential for designing rational approaches to type and track Mycoplasma genitalium as antibiotic resistance increases. It represents a first approach to its population genetics to better appreciate the role of this organism as a sexually transmitted pathogen

The pitfalls of platform comparison: DNA copy number array technologies assessed

<p>Abstract</p> <p>Background</p> <p>The accurate and high resolution mapping of DNA copy number aberrations has become an important tool by which to gain insight into the mechanisms of tumourigenesis. There are various commercially available platforms for such studies, but there remains no general consensus as to the optimal platform. There have been several previous platform comparison studies, but they have either described older technologies, used less-complex samples, or have not addressed the issue of the inherent biases in such comparisons. Here we describe a systematic comparison of data from four leading microarray technologies (the Affymetrix Genome-wide SNP 5.0 array, Agilent High-Density CGH Human 244A array, Illumina HumanCNV370-Duo DNA Analysis BeadChip, and the Nimblegen 385 K oligonucleotide array). We compare samples derived from primary breast tumours and their corresponding matched normals, well-established cancer cell lines, and HapMap individuals. By careful consideration and avoidance of potential sources of bias, we aim to provide a fair assessment of platform performance.</p> <p>Results</p> <p>By performing a theoretical assessment of the reproducibility, noise, and sensitivity of each platform, notable differences were revealed. Nimblegen exhibited between-replicate array variances an order of magnitude greater than the other three platforms, with Agilent slightly outperforming the others, and a comparison of self-self hybridizations revealed similar patterns. An assessment of the single probe power revealed that Agilent exhibits the highest sensitivity. Additionally, we performed an in-depth visual assessment of the ability of each platform to detect aberrations of varying sizes. As expected, all platforms were able to identify large aberrations in a robust manner. However, some focal amplifications and deletions were only detected in a subset of the platforms.</p> <p>Conclusion</p> <p>Although there are substantial differences in the design, density, and number of replicate probes, the comparison indicates a generally high level of concordance between platforms, despite differences in the reproducibility, noise, and sensitivity. In general, Agilent tended to be the best aCGH platform and Affymetrix, the superior SNP-CGH platform, but for specific decisions the results described herein provide a guide for platform selection and study design, and the dataset a resource for more tailored comparisons.</p

RCandy: an R package for visualizing homologous recombinations in bacterial genomes

SUMMARY: Homologous recombination is an important evolutionary process in bacteria and other prokaryotes, which increases genomic sequence diversity and can facilitate adaptation. Several methods and tools have been developed to detect genomic regions recently affected by recombination. Exploration and visualization of such recombination events can reveal valuable biological insights, but it remains challenging. Here, we present RCandy, a platform-independent R package for rapid, simple and flexible visualization of recombination events in bacterial genomes. AVAILABILITY AND IMPLEMENTATION: RCandy is an R package freely available for use under the MIT license. It is platform-independent and has been tested on Windows, Linux and MacOSX. The source code comes together with a detailed vignette available on GitHub at https://github.com/ChrispinChaguza/RCandy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

The Open-Fracture Patient Evaluation Nationwide (OPEN) study: epidemiology of open fracture care in the UK

Aims Understanding of open fracture management is skewed due to reliance on small-number lower limb, specialist unit reports and large, unfocused registry data collections. To address this, we carried out the Open Fracture Patient Evaluation Nationwide (OPEN) study, and report the demographic details and the initial steps of care for patients admitted with open fractures in the UK. Methods Any patient admitted to hospital with an open fracture between 1 June 2021 and 30 September 2021 was included, excluding phalanges and isolated hand injuries. Institutional information governance approval was obtained at the lead site and all data entered using Research Electronic Data Capture. Demographic details, injury, fracture classification, and patient dispersal were detailed. Results In total, 1,175 patients (median age 47 years (interquartile range (IQR) 29 to 65), 61.0% male (n = 717)) were admitted across 51 sites. A total of 546 patients (47.1%) were employed, 5.4% (n = 63) were diabetic, and 28.8% (n = 335) were smokers. In total, 29.0% of patients (n = 341) had more than one injury and 4.8% (n = 56) had two or more open fractures, while 51.3% of fractures (n = 637) occurred in the lower leg. Fractures sustained in vehicle incidents and collisions are common (38.8%; n = 455) and typically seen in younger patients. A simple fall (35.0%; n = 410) is common in older people. Overall, 69.8% (n = 786) of patients were admitted directly to an orthoplastic centre, 23.0% (n = 259) were transferred to an orthoplastic centre after initial management elsewhere, and 7.2% were managed outwith specialist units (n = 81). Conclusion This study describes the epidemiology of open fractures in the UK. For a decade, orthopaedic surgeons have been practicing in a guideline-driven, network system without understanding the patient features, injury characteristics, or dispersal processes of the wider population. This work will inform care pathways as the UK looks to the future of trauma networks and guidelines, and how to optimize care for patients with open fractures