Microbial profiling using metagenomic assembly

The application of second generation sequencing technology to the characterization of complex microbial communities has profoundly affected our appreciation of microbial diversity. The explosive growth of microbial sequence data has also necessitated advances in bioinformatic methods for profiling microbial communities. Data aggregation strategies should allow the relation of metagenomic sequence data to our understanding of microbial taxonomy, while also facilitating the discovery of novel taxa. For eukaryotes, a method has been established that links DNA sequences to the identification of organisms: DNA Barcoding. A similar approach has been developed for prokaryotes using target genic regions as markers for species identification and to profile communities. A key difference in these efforts is that within DNA barcoding there is a formalized framework for the evaluation of barcoding targets, whereas for prokaryotes the 16S rRNA gene target has become the de facto barcode without formal evaluation. Using the framework developed for evaluating DNA barcodes in eukaryotes, a study was undertaken to formally evaluate 16S rRNA and cpn60 as DNA barcodes for Bacteria. Both 16S rRNA and cpn60 were found to meet the criteria for DNA barcodes, with cpn60 a preferred barcode based on its superior resolution of closely related taxa. The high resolution of cpn60 enabled a method of sequence data aggregation through sequence assembly: microbial profiling using metagenomic assembly (mPUMA). The scoring of metagenomic assemblies in terms of sensitivity and specificity of the operational taxonomic units formed was used to evaluate and optimize the assembly of cpn60 barcodes. Using optimized parameters, mPUMA was demonstrated to faithfully reconstruct a synthetic community in terms of richness and abundance. To facilitate the use of mPUMA, a software package was developed and released under an open source license. The utility of mPUMA was further examined through the characterization of the epiphytic seed microbiomes of Triticum and Brassica species. A microbiome shared across both crop genera including fungi and bacteria was detected: a particularly important observation as it implies that seeds may serve as a vector for microbes that could include both pathogenic and beneficial organisms. The relative abundances of taxa identified by mPUMA were confirmed by qPCR for multiple cases of both fungal and bacterial taxa. By culturing isolates of both bacteria and fungi from the seed surfaces it was demonstrated that mPUMA faithfully assembled consensus sequences for OTUs that were 100% identical to isolated fungi and bacteria. Patterns observed in the relative abundances of the shared microbiome OTUs were used to generate the hypothesis that an Pantoea-like bacterium and an Alternaria-like fungus had an antagonistic relationship, since sequences corresponding to these organisms showed reciprocal abundance patterns on Triticum and Brassica seeds. Studies of the interactions of cultured isolates revealed fungistatic interactions that could account for their reciprocal abundances. These interactions could be directly relevant to plant health, given that Alternaria-like fungi are linked to grain spoilage in wheat, and diseases in canola. Taken together, results of this thesis demonstrate the superiority of the cpn60 universal target as a barcode for Bacteria, forming the basis for an assembly-based strategy for microbial profiling of bacterial and eukaryotic microbial communities that can lead to the discovery of novel taxa and microbial interactions

"Walking the Walk”: Promoting Competencies for Interprofessional Learning through Team Meetings—A Case Study

Background: Interprofessional learning is a key aspect of improving team-based healthcare. Core competencies for interprofessional education (IPE) activities have recently been developed, but there is a lack of guidance as to practical application. Methods and Findings: Cancer Forum is a weekly multi-professional meeting used as the case study for this report. Power was identified as a critical issue and six questions were identified as the basis for a structured reflection on the conduct of Cancer Forum. Results were then synthesised using Habermas’ delineation of learning as instrumental, normative, communicative, dramaturgical, and emancipatory. Power was a key issue in identified obstacles to inter professional learning. Leadership emerged as a cross-cutting theme and was added as a seventh question. The emancipatory potential of interprofessional learning benefited from explicit consideration of the meeting agenda to promote competencies of sharing role knowledge, teamwork and communication. Modelling of required skills fulfils a dramaturgical and normative role. Conclusions: The structured reflection tool highlighted the relationship between power and IPE competencies. It was essential to walk the walk as well as talk. The process followed provides a practical guide for using team meetings to promote interprofessional learning competencies and thereby improving patient care

Learning and change

Learning and change are intertwined, universal features of modern life. Health professional education often aims to link learning to changes in health outcomes. This thesis examines the relationship between learning, change and improvements in health outcomes. This relationship is explored in three key areas: Continuing Professional Development (CPD) for Physicians, interprofessional education and clinical leadership. The interaction between learning, change and leadership are explored through a mixture of: literature review, applied theory, theory and model development, expository writing and an auto-ethnographic case study. The theoretical approach is based on Jurgen Habermas’ theory of cognitive interests viewed through a community of practice lens. Findings were that the locus of change is a key variable distinguishing between learning theories and change management theory offers a useful perspective on the problem of transfer of learning into practice. The major division in perspective for CPD is that between the individual and organisation viewpoint. Linking professional lifelong learning to improvements in quality of care requires adoption of a variety of perspectives and careful management of the tension between self-directed learning and organisational objectives. This is aided by moving from an “update” to professional learning model of CPD. Interprofessional learning remains a challenge but team meetings provide an opportunity to address core teamwork competencies. A guided reflection tool has been developed to facilitate this. Power remains a major issue inhibiting an ideal speech situation. For leadership they key issue is that the model used in the administration aligns with that used in the clinic and a therapeutic model of leaderships is proposed. Finally the issues raised in this thesis are examined from a community of practice perspective showing that this framework offers the potential for combining individual and organisational views Linking lifelong and interprofessional learning to changes in the quality of care is a major challenge. Factors identified to facilitate this include: integrated models that build upon the learning and change management literature; an appropriate model of leadership; a community of practice perspective and an approach that considers questions not only from a purposive-technical perspective, but considers the importance of hermeneutic and emancipatory considerations

ISBAR+ a communication tool to advocate for patients

IntroductionRecognising the importance of social determinants of health is a key part of the curriculum for health practitioners. The ability to advocate on behalf of patients is a competency that demonstrates enacting this understanding in practice. Communication frameworks are used to structure difficult conversations in multiple settings, notably handover. There is no commonly accepted communication framework to structure a patient advocacy conversation.ApproachWe assembled a team with skills in patient advocacy, healthcare communication, community advocacy, education and business negotiation to identify the knowledge, skills and attitudes required and to develop a framework suitable for this purpose. We chose to adapt the ISBAR framework as an existing communication framework commonly used for handover.OutcomesISBAR+ is a framework that is based on a person-centred approach and ‘integrated negotiation’. ‘Intention and Inquiry’ involves a compassionate understanding of the patient’s position. ‘Situation’ is a succinct framing of the problem. ‘Background’ is the information required for the decision-maker to make a person-centred decision. The next step is ‘alignment’ of the priorities of the patient, practitioner and decision-maker. ‘Response’ is the proposed solution, and ‘+’ (‘plus’) is the actions taken for implementation.ConclusionsISBAR+ provides a framework for conversations advocating on behalf of patients that draws from the literature around advocacy inside and outside health. A communication framework allows the development and evaluation of interventions to teach and promote this critical function to promote person-centred care

Quantitative evaluation of bias in barcode markers derived from complex samples

PCR products have become a major commodity used to identify organisms based on polymorphism at the DNA level. One problem arising is that unbiased identification of organisms takes as working hypothesis that when DNA is extracted from a sample, a positive signal will be obtained if universal primers are used and DNA quality is suitable for PCR. As this assumption is not always correct we used a system where large differences in PCR success have been described to identify where biases appear and maybe identify solutions. Plants can be identified with at least seven independent plastid‐located loci. These differ in their degree of PCR success and how informative they are in terms of taxonomically useful sequence polymorphisms. Here we used six common plastid loci spanning 48 plant species and performed a quantitative analysis of bias at each step of the identification process. As expected we found important differences in PCR efficiency within a single species, depending on the barcoding sequence being amplified. Quantitative PCR revealed that the Ct threshold for various plastid loci, even within a single species, could exhibit greater than 2000‐fold differences in DNA quantity after amplification. We then performed Next Generation Sequencing experiments in nine species using equal quantities of three plastid‐based primers and equally‐mixed quantities of DNA from multiple species. The result was significantly biased towards species and specific loci even when using adaptor‐specific primers. Our results caution that Next‐Generation Sequencing projects may suffer dramatic bias, arising largely during DNA amplification steps. Moreover, that amplification‐based Next Generation Sequencing technologies exhibit additional bias despite using adaptor‐specific primers, indicating that amplification success depends on the DNA fragment. As such, while qualitative analysis of unknown samples are prone to false negative results if a combination of widely‐successful amplicons are not used, quantitative results should be considered highly suspect, even if all species in the starting sample are known.This work was funded by the Comunidad Autónoma de la Región de Murcia Project “Molecular markers in coservation and management of the flora of Murcia Region”

Quantitative evaluation of bias in PCR amplification and Next Generation Sequencing derived from metabarcoding samples

Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to Next-Generation Sequencing. End-point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2000-fold differences in DNA quantity after amplification. Next Generation Sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of Q-PCR amplification.This work was funded by the Comunidad Autónoma de la Región de Murcia Project “Molecular markers in conservation and management of the flora of Murcia Region”. This work was published in Analytical and Bioanalytical Chemistry (407(7): 1841-8

Wheat EST resources for functional genomics of abiotic stress

BACKGROUND: Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project. RESULTS: We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology. CONCLUSION: We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals