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Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND ( G enome-derived R ibosomal O pero n D atabase), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.

Mechanisms underlying the disruption of self-tolerance in acquired autoimmunity remain unclear. Immunoglobulin A (IgA) nephropathy is an acquired autoimmune disease where deglycosylated IgA1 (IgA subclass 1) auto-antigens are recognized by IgG auto-antibodies, forming immune complexes that are deposited in the kidneys, leading to glomerulonephritis. In the intestinal microbiota of patients with IgA nephropathy, there was increased relative abundance of mucin-degrading bacteria, including Akkermansia muciniphila . IgA1 was deglycosylated by A. muciniphila both in vitro and in the intestinal lumen of mice. This generated neo-epitopes that were recognized by autoreactive IgG from the sera of patients with IgA nephropathy. Mice expressing human IgA1 and the human Fc α receptor I (α1 KI -CD89 tg ) that underwent intestinal colonization by A. muciniphila developed an aggravated IgA nephropathy phenotype. After deglycosylation of IgA1 by A. muciniphila in the mouse gut lumen, IgA1 crossed the intestinal epithelium into the circulation by retrotranscytosis and became deposited in the glomeruli of mouse kidneys. Human α-defensins-a risk locus for IgA nephropathy-inhibited growth of A. muciniphila in vitro. A negative correlation observed between stool concentration of α-defensin 6 and quantity of A. muciniphila in the guts of control participants was lost in patients with IgA nephropathy. This study demonstrates that gut microbiota dysbiosis contributes to generation of auto-antigens in patients with IgA nephropathy and in a mouse model of this disease.

A systematic approach to collect, peruse, and summarize the available information relating to the potential benefits of consuming dietary microbes was pursued in this scoping review. This review focused on the research endpoints, experimental designs, and microbial exposure in experimental as well as observational research work. Using a structured- set of keywords, scientific databases were systematically searched to retrieve publications reporting outcomes pertaining to the use of dietary microbes in healthy, nonpatient populations. Searches were further tailored to focus on eight different health categories, namely, "antibiotic associated diarrhoea" (AAD), "gastrointestinal health" (GIH), "immunological health" (ImH), "cardiovascular health and metabolic syndrome" (CvHMS), "cancer prevention" (CanPr), "respiratory health" (ReH), "weight management" (WtMgt), and "urogenital health" (UrGH). Quality of evidence available in each publication was assessed using the Jadad scoring system. The search yielded 228 relevant publications describing 282 experimental cases comprising 62 research endpoints overall. A microbial dose of ≥ 2 × 10 9 $\ge 2\times 10^9$ CFU.day -1 was associated with non-negative reported outcomes. Older population groups with a median age of 39 years were associated with positive outcomes. More high-quality research is required investigating the role of dietary microbes in maintaining general health, particularly in the health categories of UrGH, WtMgt, and CanPr., (© 2024 The Authors. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

Chlorate has become a concern in the food and beverage sector, related to chlorine sanitizers in industrial food production and water treatment. It is of particular concern to regulatory bodies due to the negative health effects of chlorate exposure. This study investigated the fate of chlorate in raw milk and isolated bacterial strains of interest responsible for chlorate breakdown. Unpasteurized milk was demonstrated to have a chlorate-reducing capacity, breaking down enriched chlorate to undetectable levels in 11 days. Further enrichment and isolation using conditions specific to chlorate-reducing bacteria successfully isolated three distinct strains of Hafnia paralvei . Chlorate-reducing bacteria were observed to grow in a chlorate-enriched medium with lactate as an electron donor. All isolated strains were demonstrated to reduce chlorate in liquid medium; however, the exact mechanism of chlorate degradation was not definitively identified in this study.

Introduction: It has been hypothesised that the regular consumption of safe, live microbes confers health-promoting attributes, including the prevention of disease. To address this hypothesis, we propose a scoping review approach that will systematically assess the large corpus of relevant literature that is now available on this research topic. This article outlines a protocol for a scoping review of published studies on interventions with live microbes in non-patient populations across eight health categories. The scoping review aims to catalogue types of interventions, measured outcomes, dosages, effectiveness, as well as current research gaps., Methods and Analysis: The scoping review will follow the six-staged protocol as proposed by Arksey and O'Malley and will include the following stages: defining the research questions (stage 1); defining the eligibility criteria and finalising search strategy (stage 2); selection of studies based on the eligibility criteria (stage 3); development of a data extraction framework and charting of data (stage 4); aggregation of results and summarisation of findings (stage 5); and the optional consultation with stakeholders (stage 6), which will not be performed., Ethics and Dissemination: Since the scoping review synthesises information from existing literature, no separate ethical approval is required. The findings of the scoping review will be communicated for publication to an open-access, peer-reviewed scientific journal, presented at relevant conferences, and disseminated at future workshops with all relevant data and documents being available online through the Open Science Framework (https://osf.io/kvhe7)., Competing Interests: Competing interests: PDC has received funding from Danone, PepsiCo and PrecisionBiotics Group and is sponsored by PepsiCo, H&H, National Dairy Council (USA) to contribute to conferences/meetings. He is also a co-founder and CTO of SeqBiome., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Peri-hilar cholangiocarcinoma (pCCA) is chemorefractory and limited genomic analyses have been undertaken in Western idiopathic disease. We undertook comprehensive genomic analyses of a U.K. idiopathic pCCA cohort to characterize its mutational profile and identify new targets. Whole exome and targeted DNA sequencing was performed on forty-two resected pCCA tumors and normal bile ducts, with Gene Set Enrichment Analysis (GSEA) using one-tailed testing to generate false discovery rates (FDR). 60% of patients harbored one cancer-associated mutation, with two mutations in 20%. High frequency somatic mutations in genes not typically associated with cholangiocarcinoma included mTOR, ABL1 and NOTCH1. We identified non-synonymous mutation (p.Glu38del) in MAP3K9 in ten tumors, associated with increased peri-vascular invasion (Fisher's exact, p < 0.018). Mutation-enriched pathways were primarily immunological, including innate Dectin-2 (FDR 0.001) and adaptive T-cell receptor pathways including PD-1 (FDR 0.007), CD4 phosphorylation (FDR 0.009) and ZAP70 translocation (FDR 0.009), with overlapping HLA genes. We observed cancer-associated mutations in over half of our patients. Many of these mutations are not typically associated with cholangiocarcinoma yet may increase eligibility for contemporary targeted trials. We also identified a targetable MAP3K9 mutation, in addition to oncogenic and immunological pathways hitherto not described in any cholangiocarcinoma subtype., (© 2023. The Author(s).)

Objectives: Juvenile-onset systemic lupus erythematosus (jSLE) affects 15-20% of lupus patients. Clinical heterogeneity between racial groups, age groups and individual patients suggests variable pathophysiology. This study aimed to identify highly penetrant damaging mutations in genes associated with SLE/SLE-like disease in a large national cohort (UK JSLE Cohort Study) and compare demographic, clinical and laboratory features in patient sub-cohorts with 'genetic' SLE vs remaining SLE patients., Methods: Based on a sequencing panel designed in 2018, target enrichment and next-generation sequencing were performed in 348 patients to identify damaging gene variants. Findings were integrated with demographic, clinical and treatment related datasets., Results: Damaging gene variants were identified in ∼3.5% of jSLE patients. When compared with the remaining cohort, 'genetic' SLE affected younger children and more Black African/Caribbean patients. 'Genetic' SLE patients exhibited less organ involvement and damage, and neuropsychiatric involvement developed over time. Less aggressive first line treatment was chosen in 'genetic' SLE patients, but more second and third line agents were used. 'Genetic' SLE associated with anti-dsDNA antibody positivity at diagnosis and reduced ANA, anti-LA and anti-Sm antibody positivity at last visit., Conclusion: Approximately 3.5% of jSLE patients present damaging gene variants associated with younger age at onset, and distinct clinical features. As less commonly observed after treatment induction, in 'genetic' SLE, autoantibody positivity may be the result of tissue damage and explain reduced immune complex-mediated renal and haematological involvement. Routine sequencing could allow for patient stratification, risk assessment and target-directed treatment, thereby increasing efficacy and reducing toxicity., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

The microbial communities present within fermented foods are diverse and dynamic, producing a variety of metabolites responsible for the fermentation processes, imparting characteristic organoleptic qualities and health-promoting traits, and maintaining microbiological safety of fermented foods. In this context, it is crucial to study these microbial communities to characterise fermented foods and the production processes involved. High Throughput Sequencing (HTS)-based methods such as metagenomics enable microbial community studies through amplicon and shotgun sequencing approaches. As the field constantly develops, sequencing technologies are becoming more accessible, affordable and accurate with a further shift from short read to long read sequencing being observed. Metagenomics is enjoying wide-spread application in fermented food studies and in recent years is also being employed in concert with synthetic biology techniques to help tackle problems with the large amounts of waste generated in the food sector. This review presents an introduction to current sequencing technologies and the benefits of their application in fermented foods.

Bacterial infections of livestock threaten the sustainability of agriculture and public health through production losses and contamination of food products. While prophylactic and therapeutic application of antibiotics has been successful in managing such infections, the evolution and spread of antibiotic-resistant strains along the food chain and in the environment necessitates the development of alternative or adjunct preventive and/or therapeutic strategies. Additionally, the growing consumer preference for "greener" antibiotic-free food products has reinforced the need for novel and safer approaches to controlling bacterial infections. The use of bacteriophages (phages), which can target and kill bacteria, are increasingly considered as a suitable measure to reduce bacterial infections and contamination in the food industry. This review primarily elaborates on the recent veterinary applications of phages and discusses their merits and limitations. Furthermore, using Streptococcus suis as a model, we describe the prevalence of prophages and the anti-viral defence arsenal in the genome of the pathogen as a means to define the genetic building blocks that are available for the (synthetic) development of phage-based treatments. The data and approach described herein may provide a framework for the development of therapeutics against an array of bacterial pathogens.

This paper reports on research exploring the academic workload and performance practices of Australian universities. This research has identified a suite of activities associated with teaching, research and service, each with an associated time value (allocation). This led to the development of the academic workload estimation tool (AWET). In 2020, to validate the findings, we contacted academics willing to participate further and conducted interviews. We used the AWET to estimate workload for each individual for the previous year and compared it to the workload allocated according to their institutional workload model. Discrepancies were then discussed to ascertain to what extent the AWET was able to capture their work. In general, the participants thought the AWET provided a more realistic estimate of their actual work and highlighted how much is underestimated or unaccounted for by the workload models used within their institutions. It also showed how academic performance policies, focussed primarily on research output, disadvantaged many individuals because they ignored or minimised many scholarly, teaching and service-related tasks inherent in the academic role. Overall, the findings showed the AWET was a useful tool to discuss academic work and assisted them to better capture the complexity and extent of what they did. We offer the AWET as a validated approach for academics to estimate their workload in a holistic and transparent manner. We suggest its implementation institution-wide, along with an aligned performance policy, will facilitate negotiation of reasonable performance expectations. This will rebuild trust in the processes and improve a university's effectiveness., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s) 2022.)

In recent years, there has been a global resurgence of public interest in fermented foods. In parallel, there have been several new studies that associate the consumption of fermented foods with a variety of beneficial impacts. These combined developments have led to a renewed focus in research and innovation vis-à-vis fermented foods, particularly traditional fermented foods, with an aim to harness this information to develop novel fermented foodstuffs and ingredients and make them available in the market. Consequently, an ever greater and more diverse array of fermented foods, including functional fermented foods with health benefits, are becoming available for public consumption in global markets, with the number expected to grow substantially in the coming decade. This rapidly expanding portfolio of commercially available fermented foods has in turn required an evolution in the corresponding global regulatory frameworks. Due to the innovative and emerging nature of these foods, combined with historical differences in regulator approaches, significant disharmony exists across these frameworks, with individual nations and organizations often adopting unique approaches relating to the establishment of standards and specifications. In this review, we provide an overview of the current regulatory frameworks for a diversity of fermented foods across multiple jurisdictions, with special emphasis on differences in legislative structures and approaches, regulatory harmonization, and current legislative limitations. Overall, the review provides important perspective and context in relation to current global fermented food regulatory practices with possible directions and recommendations for future legislative efforts., Competing Interests: PC has received funding from Danone, PepsiCo and PrecisionBiotics Group and is sponsored by PepsiCo, H&H, National Dairy Council (USA) to contribute to conferences/meetings. He is also a co-founder and CTO of SeqBiome. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mukherjee, Gómez-Sala, O'Connor, Kenny and Cotter.)

Internationally, much has changed in the governance of universities since the adoption of corporate management approaches. A strong focus on efficiency, productivity and accountability arising from these approaches has been well documented in the literature. Reductions in government funding have caused universities to become more competitive and entrepreneurial. However, little is known about the impacts of these changes on the working lives of individual academics. This paper is part of an ongoing study exploring the lived experiences of 2526 Australian academics who responded to a national questionnaire. This paper builds on earlier work by holistically drawing together the earlier findings which separately analysed the teaching, research and administration/service aspects of their work. In examining the effectiveness of universities through the ability of their academics to undertake their roles, we found the voices of academics that need to be heard in the development and implementation of key policies, such as academic workload and performance, to preserve the essentially self-managed nature of their work. By combining the learning from the project through the literature review, the statistical analysis and themes from the open-ended questions, we developed a set of principles to underpin these policies in universities. These principles can guide universities to shift towards a more collaborative working relationship with academics, based on trust, and actively encourage them to play be more active in institutional decision-making, especially in relation to policies that directly affect their work. These results have implications for improving the productivity of academics and the institutions in which they work., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

Background and Purpose: Synthetic diamond detectors offer real time measurement of dose in radiotherapy applications which require high spatial resolution. Additional considerations and corrections are required for measurements where the diamond detector is orientated at various angles to the incident beam. This study investigated diamond detectors for end-to-end testing of Stereotactic Body Radiotherapy (SBRT) and Stereotactic Radiosurgery (SRS) in the context of dosimetry audits., Material and Methods: Seven individual diamond detectors were investigated and compared with respect to warm up stability, dose-rate dependence, linearity, detector shadowing, energy response, cross-calibration, angular dependence and positional sensitivity in SBRT and SRS., Results: Large variation in the cross calibration factors was found between the seven individual detectors. For each detector, the energy dependence in the cross calibration factor was on average <0.6% across the beam qualities investigated (Co-60 Gamma Knife, and MV beams with TPR 20,10 0.684-0.733). The angular corrections for individual fields were up to 5%, and varied with field size. However, the average angular dependence for all fields in a typical SRS treatment delivery was <1%. The overall measurement uncertainty was 3.6% and 3.1% (2σ) for an SRS and SBRT treatment plan respectively., Conclusion: Synthetic diamond detectors were found to be reliable and robust for end-to-end dosimetry in SBRT and SRS applications. Orientation of the detector relative to the beam axis is an important consideration, as significant corrections are required for angular dependence., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2021 Published by Elsevier B.V. on behalf of European Society of Radiotherapy & Oncology.)

Population growth, the impacts of climate change, and the need for greater water security have made the reuse of wastewater, including potable use, increasingly desirable. As interest in potable reuse of wastewater increases, a variety of processes have been proposed for advanced water treatment following conventional wastewater treatment. In all cases, the operation and performance of advanced water treatment facilities (AWTFs) is improved when the treated wastewater feed is of the highest quality that can be achieved and the advanced water treatment (AWT) processes are operated at a constant flow. One proven method of optimizing the performance of wastewater treatment facilities (WWTFs) is constant flow operation with no extraneous return flows other than internal process recycle flows, such as return settled solids. A number of approaches can be used to achieve constant flow including flow equalization, divided treatment trains, and satellite treatment. The ways in which constant flow wastewater treatment benefits both WWTFs as well as the AWTFs are considered with special emphasis on the ability to achieve predictable log removal credits (LRCs) for specific microorganisms. Actual performance data from constant flow WWTFs are used to illustrate how LRCs are determined. PRACTITIONER POINTS: Constant flow WWTFs should be considered to produce the highest quality secondary effluent for AWT. Flow equalization, divided treatment trains, and satellite treatment can be used to achieve constant flow to optimize wastewater treatment in small and medium size WWTFs. Flow equalization can be used to maximize the amount of wastewater that can be recovered for potable reuse. Important benefits of constant flow for wastewater treatment facilities include economic and operational savings, stable and predictable treatment performance, energy savings, ability to optimize performance for the removal of specific constituents, and the ability to assign pathogen log removal credits (LRCs). Important benefits of constant flow and optimized WWT for AWTFs include economic and operational savings; less pretreatment needed, including energy and chemical usage; elimination of the need to cycle treatment processes; and added factor of safety with respect to the required pathogen LRCs. In large WWTFs, constant flow for AWTFs will typically be achieved by effluent diversion; depending on the effluent quality additional pretreatment may be needed. The design and implementation of WWTFs and AWTFs for potable reuse should be integrated for optimal performance and protection of public health., (© 2021 The Authors. Water Environment Research published by Wiley Periodicals LLC on behalf of Water Environment Federation.)

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 μM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

Alterations in the human microbiome have been observed in a variety of conditions such as asthma, gingivitis, dermatitis and cancer, and much remains to be learned about the links between the microbiome and human health. The fusion of artificial intelligence with rich microbiome datasets can offer an improved understanding of the microbiome's role in human health. To gain actionable insights it is essential to consider both the predictive power and the transparency of the models by providing explanations for the predictions. We combine the collection of leg skin microbiome samples from two healthy cohorts of women with the application of an explainable artificial intelligence (EAI) approach that provides accurate predictions of phenotypes with explanations. The explanations are expressed in terms of variations in the relative abundance of key microbes that drive the predictions. We predict skin hydration, subject's age, pre/post-menopausal status and smoking status from the leg skin microbiome. The changes in microbial composition linked to skin hydration can accelerate the development of personalized treatments for healthy skin, while those associated with age may offer insights into the skin aging process. The leg microbiome signatures associated with smoking and menopausal status are consistent with previous findings from oral/respiratory tract microbiomes and vaginal/gut microbiomes respectively. This suggests that easily accessible microbiome samples could be used to investigate health-related phenotypes, offering potential for non-invasive diagnosis and condition monitoring. Our EAI approach sets the stage for new work focused on understanding the complex relationships between microbial communities and phenotypes. Our approach can be applied to predict any condition from microbiome samples and has the potential to accelerate the development of microbiome-based personalized therapeutics and non-invasive diagnostics.

The etiology of Crohn's disease (CD) is multifactorial. Bacterial and fungal microbiota are involved in the onset and/or progression of the disease. A bacterial dysbiosis in CD patients is accepted; however, less is known about the mycobiome and the relationships between the two communities. We investigated the interkingdom relationships, their metabolic consequences, and the changes in the fungal community during relapse and remission in CD.Two cohorts were evaluated: a British cohort (n = 63) comprising CD and ulcerative colitis patients, and controls. The fungal and bacterial communities of biopsy and fecal samples were analyzed, with the fecal volatiles; datasets were also integrated; and a Dutch cohort (n = 41) comprising CD patients and healthy controls was analyzed for stability of the gut mycobiome.A dysbiosis of the bacterial community was observed in biopsies and stool. Results suggest Bacteroides is likely key in CD and may modulate Candida colonization. A dysbiosis of the fungal community was observed only in the Dutch cohort; Malassezia and Candida were increased in patients taking immunosuppressants. Longitudinal analysis showed an increase in Cyberlindnera in relapse. Saccharomyces was dominant in all fecal samples, but not in biopsies, some of which did not yield fungal reads; amino acid degradation was the main metabolic change associated with CD and both bacteria and fungi might be implicated.We have shown that Bacteroides and yeasts may play a role in CD; understanding their role and relationship in the disease would shed new light on the development and treatment of CD.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX , most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX , as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1 , and SF3B1 and EIF1AX , respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1 . NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.

Background: Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease., Methods: We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993-1996; n = 293] and IID2 [isolates from 2008-2009; n = 93]), the INTEGRATE project [isolates from 2016-2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163]., Results: There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation., Discussion: Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation., Competing Interests: The authors have declared that no competing interests exist.

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50 % pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple 'omic' strategies, combining Illumina and Pacific Biosciences Single-Molecule Real-Time DNA sequencing and annotation, stranded RNA sequencing and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53 and 74 % of the Sodalis transcriptome remains active in cell-free culture. The mean sense transcription from coding domain sequences (CDSs) is four times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40 % of the 2729 genes in the core genome, suggesting that they are stable and/or that Sodalis is a recent introduction across the genus Glossina as a facultative symbiont. These data shed further light on the importance of transcriptional and translational control in deciphering host-microbe interactions. The combination of genomics, transcriptomics and proteomics gives a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

The endocardium is the endothelial component of the vertebrate heart and plays a key role in heart development. Where, when, and how the endocardium segregates during embryogenesis have remained largely unknown, however. We now show that Nkx2-5 + cardiac progenitor cells (CPCs) that express the Sry-type HMG box gene Sox17 from embryonic day (E) 7.5 to E8.5 specifically differentiate into the endocardium in mouse embryos. Although Sox17 is not essential or sufficient for endocardium fate, it can bias the fate of CPCs toward the endocardium. On the other hand, Sox17 expression in the endocardium is required for heart development. Deletion of Sox17 specifically in the mesoderm markedly impaired endocardium development with regard to cell proliferation and behavior. The proliferation of cardiomyocytes, ventricular trabeculation, and myocardium thickening were also impaired in a non-cell-autonomous manner in the Sox17 mutant, likely as a consequence of down-regulation of NOTCH signaling. An unknown signal, regulated by Sox17 and required for nurturing of the myocardium, is responsible for the reduction in NOTCH-related genes in the mutant embryos. Our results thus provide insight into differentiation of the endocardium and its role in heart development.

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads-the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process., Competing Interests: The authors have declared that no competing interests exist.

Soil biota accounts for ~25% of global biodiversity and is vital to nutrient cycling and primary production. There is growing momentum to study total belowground biodiversity across large ecological scales to understand how habitat and soil properties shape belowground communities. Microbial and animal components of belowground communities follow divergent responses to soil properties and land use intensification; however, it is unclear whether this extends across heterogeneous ecosystems. Here, a national-scale metabarcoding analysis of 436 locations across 7 different temperate ecosystems shows that belowground animal and microbial (bacteria, archaea, fungi, and protists) richness follow divergent trends, whereas β-diversity does not. Animal richness is governed by intensive land use and unaffected by soil properties, while microbial richness was driven by environmental properties across land uses. Our findings demonstrate that established divergent patterns of belowground microbial and animal diversity are consistent across heterogeneous land uses and are detectable using a standardised metabarcoding approach.

Biologic therapies have shown high efficacy in psoriasis, but individual response varies and is poorly understood. To inform biomarker discovery in the Psoriasis Stratification to Optimise Relevant Therapy (i.e., PSORT) study, we evaluated a comprehensive array of omics platforms across three time points and multiple tissues in a pilot investigation of 10 patients with severe psoriasis, treated with the tumor necrosis factor (TNF) inhibitor, etanercept. We used RNA sequencing to analyze mRNA and small RNA transcriptome in blood, lesional and nonlesional skin, and the SOMAscan platform to investigate the serum proteome. Using an integrative systems biology approach, we identified signals of treatment response in genes and pathways associated with TNF signaling, psoriasis pathology, and the major histocompatibility complex region. We found association between clinical response and TNF-regulated genes in blood and skin. Using a combination of differential expression testing, upstream regulator analysis, clustering techniques, and predictive modeling, we show that baseline samples are indicative of patient response to biologic therapies, including signals in blood, which have traditionally been considered unreliable for inference in dermatology. In conclusion, our pilot study provides both an analytical framework and empirical basis to estimate power for larger studies, specifically the ongoing PSORT study, which we show as powered for biomarker discovery and patient stratification., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Wheat has been domesticated into a large number of agricultural environments and has the ability to adapt to diverse environments. To understand this process, we survey genotype, repeat content, and DNA methylation across a bread wheat landrace collection representing global genetic diversity. We identify independent variation in methylation, genotype, and transposon copy number. We show that these, so far unexploited, sources of variation have had a significant impact on the wheat genome and that ancestral methylation states become preferentially "hard coded" as single nucleotide polymorphisms (SNPs) via 5-methylcytosine deamination. These mechanisms also drive local adaption, impacting important traits such as heading date and salt tolerance. Methylation and transposon diversity could therefore be used alongside SNP-based markers for breeding., (© 2018 Gardiner et al.; Published by Cold Spring Harbor Laboratory Press.)

Marine sediments are important sites for global biogeochemical cycling, mediated by macrofauna and microalgae. However, it is the microorganisms that drive these key processes. There is strong evidence that coastal benthic habitats will be affected by changing environmental variables (rising temperature, elevated CO 2 ), and research has generally focused on the impact on macrofaunal biodiversity and ecosystem services. Despite their importance, there is less understanding of how microbial community assemblages will respond to environmental changes. In this study, a manipulative mesocosm experiment was employed, using next-generation sequencing to assess changes in microbial communities under future environmental change scenarios. Illumina sequencing generated over 11 million 16S rRNA gene sequences (using a primer set biased toward bacteria) and revealed Bacteroidetes and Proteobacteria dominated the total bacterial community of sediment samples. In this study, the sequencing coverage and depth revealed clear changes in species abundance within some phyla. Bacterial community composition was correlated with simulated environmental conditions, and species level community composition was significantly influenced by the mean temperature of the environmental regime ( p = 0.002), but not by variation in CO 2 or diurnal temperature variation. Species level changes with increasing mean temperature corresponded with changes in NH 4 concentration, suggesting there is no functional redundancy in microbial communities for nitrogen cycling. Marine coastal biogeochemical cycling under future environmental conditions is likely to be driven by changes in nutrient availability as a direct result of microbial activity.

Background and Purpose: Independent dosimetry audits improve quality and safety of radiation therapy. This work reports on design and findings of a comprehensive 3D conformal radiotherapy (3D-CRT) Level III audit., Materials and Methods: The audit was conducted as onsite audit using an anthropomorphic thorax phantom in an end-to-end test by the Australian Clinical Dosimetry Service (ACDS). Absolute dose point measurements were performed with Farmer-type ionization chambers. The audited treatment plans included open and half blocked fields, wedges and lung inhomogeneities. Audit results were determined as Pass Optimal Level (deviations within 3.3%), Pass Action Level (greater than 3.3% but within 5%) and Out of Tolerance (beyond 5%), as well as Reported Not Scored (RNS). The audit has been performed between July 2012 and January 2018 on 94 occasions, covering approximately 90% of all Australian facilities., Results: The audit pass rate was 87% (53% optimal). Fifty recommendations were given, mainly related to planning system commissioning. Dose overestimation behind low density inhomogeneities by the analytical anisotropic algorithm (AAA) was identified across facilities and found to extend to beam setups which resemble a typical breast cancer treatment beam placement. RNS measurements inside lung showed a variation in the opposite direction: AAA under-dosed a target beyond lung and over-dosed the lung upstream and downstream of the target. Results also highlighted shortcomings of some superposition and convolution algorithms in modelling large angle wedges., Conclusions: This audit showed that 3D-CRT dosimetry audits remain relevant and can identify fundamental global and local problems that also affect advanced treatments., (Crown Copyright © 2018 Published by Elsevier B.V. on behalf of European Society of Radiotherapy & Oncology.)

Background: Bread wheat has a large complex genome that makes whole genome resequencing costly. Therefore, genome complexity reduction techniques such as sequence capture make re-sequencing cost effective. With a high-quality draft wheat genome now available it is possible to design capture probe sets and to use them to accurately genotype and anchor SNPs to the genome. Furthermore, in addition to genetic variation, epigenetic variation provides a source of natural variation contributing to changes in gene expression and phenotype that can be profiled at the base pair level using sequence capture coupled with bisulphite treatment. Here, we present a new 12 Mbp wheat capture probe set, that allows both the profiling of genotype and methylation from the same DNA sample. Furthermore, we present a method, based on Agilent SureSelect Methyl-Seq, that will use a single capture assay as a starting point to allow both DNA sequencing and methyl-seq., Results: Our method uses a single capture assay that is sequentially split and used for both DNA sequencing and methyl-seq. The resultant genotype and epi-type data is highly comparable in terms of coverage and SNP/methylation site identification to that generated from separate captures for DNA sequencing and methyl-seq. Furthermore, by defining SNP frequencies in a diverse landrace from the Watkins collection we highlight the importance of having genotype data to prevent false positive methylation calls. Finally, we present the design of a new 12 Mbp wheat capture and demonstrate its successful application to re-sequence wheat., Conclusions: We present a cost-effective method for performing both DNA sequencing and methyl-seq from a single capture reaction thus reducing reagent costs, sample preparation time and DNA requirements for these complementary analyses.

Intraspecific diversity promotes evolutionary change, and when partitioned among geographic regions or habitats can form the basis for speciation. Marine species live in an environment that can provide as much scope for diversification in the vertical as in the horizontal dimension. Understanding the relevant mechanisms will contribute significantly to our understanding of eco-evolutionary processes and effective biodiversity conservation. Here, we provide an annotated genome assembly for the deep-sea fish Coryphaenoides rupestris and re-sequencing data to show that differentiation at non-synonymous sites in functional loci distinguishes individuals living at different depths, independent of horizontal spatial distance. Our data indicate disruptive selection at these loci; however, we find no clear evidence for differentiation at neutral loci that may indicate assortative mating. We propose that individuals with distinct genotypes at relevant loci segregate by depth as they mature (supported by survey data), which may be associated with ecotype differentiation linked to distinct phenotypic requirements at different depths.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch's postulates to address the role of microbial communities in disease.

Aim: The aim of this work was to design and evaluate a software tool for analysis of a patient's respiration, with the goal of optimizing the effectiveness of motion management techniques during radiotherapy imaging and treatment., Materials and Methods: A software tool which analyses patient respiratory data files (.vxp files) created by the Varian Real-Time Position Management System (RPM) was developed to analyse patient respiratory data. The software, called RespAnalysis, was created in MATLAB and provides four modules, one each for determining respiration characteristics, providing breathing coaching (biofeedback training), comparing pre and post-training characteristics and performing a fraction-by-fraction assessment. The modules analyse respiratory traces to determine signal characteristics and specifically use a Sample Entropy algorithm as the key means to quantify breathing irregularity. Simulated respiratory signals, as well as 91 patient RPM traces were analysed with RespAnalysis to test the viability of using the Sample Entropy for predicting breathing regularity., Results: Retrospective assessment of patient data demonstrated that the Sample Entropy metric was a predictor of periodic irregularity in respiration data, however, it was found to be insensitive to amplitude variation. Additional waveform statistics assessing the distribution of signal amplitudes over time coupled with Sample Entropy method were found to be useful in assessing breathing regularity., Conclusions: The RespAnalysis software tool presented in this work uses the Sample Entropy method to analyse patient respiratory data recorded for motion management purposes in radiation therapy. This is applicable during treatment simulation and during subsequent treatment fractions, providing a way to quantify breathing irregularity, as well as assess the need for breathing coaching. It was demonstrated that the Sample Entropy metric was correlated to the irregularity of the patient's respiratory motion in terms of periodicity, whilst other metrics, such as percentage deviation of inhale/exhale peak positions provided insight into respiratory amplitude regularity., (Copyright © 2017 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.)

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

Purpose: "Ice-mile" swimming presents significant physiological challenges and potential safety issues, but few data are available. This study examined deep body temperature (BT), respiratory rate (RR), and swim performance in 2 swimmers completing an ice-mile swim of 1 mile (1600 m) in water less than 5°C., Methods: Two male cold-water-habituated swimmers completed a 1-mile lake swim in 3.9°C water. For comparative purposes, they completed an indoor 1-mile swim in 28.1°C water. The Equivital physiological monitoring system was used to record BT and RR before, during, and after each swim. Total time to complete the swims and 400-m splits were recorded., Results: One swimmer became hypothermic after 27 min while swimming, reaching BT of 33.7°C at swim's end. On exiting the water the swimmers experienced large BT after-drops of -3.6°C and -2.4°C, reaching low points of 33.2°C and 31.3°C 38 and 23 min postswim, respectively. Respiratory rate and swim pace decreased over the course of the ice-mile swim for both swimmers. Swim pace for 1 swimmer declined sharply in the final 400-m lap of the ice mile when he was hypothermic. Both swimmers remained hypothermic 60 min postswim (34.2°C and 33.4°C)., Conclusion: Ice-mile swimmers may become hypothermic while swimming, and the postswim BT after drop may expose them to dangerous levels of hypothermia. Pace and RR should be monitored as proxies for a swimmer's physiological state. Postswim recovery should also be monitored for hypothermia for at least 1 h.

The Ebola virus (EBOV) variant Makona (which emerged in 2013) was the causative agent of the largest outbreak of Ebola Virus Disease recorded. Differences in virus-host interactions between viral variants have potential consequences for transmission, disease severity and mortality. A detailed profile of the cellular changes induced by the Makona variant compared with other Ebola virus variants was lacking. In this study, A549 cells, a human cell line with a robust innate response, were infected with the Makona variant or with the Ecran variant originating from the 1976 outbreak in Central Africa. The abundance of viral and cellular mRNA transcripts was profiled using RNASeq and differential gene expression analysis performed. Differences in effects of each virus on the expression of interferon-stimulated genes were also investigated in A549 NPro cells where the type 1 interferon response had been attenuated. Cellular transcriptomic changes were compared with those induced by human respiratory syncytial virus (HRSV), a virus with a similar genome organisation and replication strategy to EBOV. Pathway and gene ontology analysis revealed differential expression of functionally important genes; including genes involved in the inflammatory response, cell proliferation, leukocyte extravasation and cholesterol biosynthesis. Whilst there was overlap with HRSV, there was unique commonality to the EBOV variants.

Background: In 2014, Western Africa experienced an unanticipated explosion of Ebola virus infections. What distinguishes fatal from non-fatal outcomes remains largely unknown, yet is key to optimising personalised treatment strategies. We used transcriptome data for peripheral blood taken from infected and convalescent recovering patients to identify early stage host factors that are associated with acute illness and those that differentiate patient survival from fatality., Results: The data demonstrate that individuals who succumbed to the disease show stronger upregulation of interferon signalling and acute phase responses compared to survivors during the acute phase of infection. Particularly notable is the strong upregulation of albumin and fibrinogen genes, which suggest significant liver pathology. Cell subtype prediction using messenger RNA expression patterns indicated that NK-cell populations increase in patients who survive infection. By selecting genes whose expression properties discriminated between fatal cases and survivors, we identify a small panel of responding genes that act as strong predictors of patient outcome, independent of viral load., Conclusions: Transcriptomic analysis of the host response to pathogen infection using blood samples taken during an outbreak situation can provide multiple levels of information on both disease state and mechanisms of pathogenesis. Host biomarkers were identified that provide high predictive value under conditions where other predictors, such as viral load, are poor prognostic indicators. The data suggested that rapid analysis of the host response to infection in an outbreak situation can provide valuable information to guide an understanding of disease outcome and mechanisms of disease.

In radiation therapy, calculation of dose within the patient contains inherent uncertainties, inaccuracies, limitations, and the potential for random error. Thus, point dose-independent verification of such calculations is a well-established process, with published data to support the setting of both action levels and tolerances. Mobius3D takes this process one step further with a full independent calculation of patient dose and comparisons of clinical parameters such as mean target dose and voxel-by-voxel gamma analysis. There is currently no published data to directly inform tolerance levels for such parameters, and therefore this work presents a database of 1000 Mobius3D results to fill this gap. The data are tested for normality using a normal probability plot and found to fit this distribution for three sub groups of data; Eclipse, iPlan and the treatment site Lung. The mean (μ) and standard deviation (σ) of these sub groups is used to set action levels and tolerances at μ ± 2σ and μ ± 3σ, respectively. A global (3%, 3 mm) gamma tolerance is set at 88.5%. The mean target dose tolerance for Eclipse data is the narrowest at ± 3%, whilst iPlan and Lung have a range of -5.0 to 2.2% and -1.8 to 5.0%, respectively. With these limits in place, future results failing the action level or tolerance will fall within the worst 5% and 1% of historical results and an informed decision can be made regarding remedial action prior to treatment., (© 2016 The Authors. Journal of Applied Clinical Medical Physics published by Wiley Periodicals, Inc. on behalf of American Association of Physicists in Medicine.)

Unlabelled: Human respiratory syncytial virus (HRSV) is a major cause of serious respiratory tract infection. Treatment options include administration of ribavirin, a purine analog, although the mechanism of its anti-HRSV activity is unknown. We used transcriptome sequencing (RNA-seq) to investigate the genome mutation frequency and viral mRNA accumulation in HRSV-infected cells that were left untreated or treated with ribavirin. In the absence of ribavirin, HRSV-specific transcripts accounted for up to one-third of total RNA reads from the infected-cell RNA population. Ribavirin treatment resulted in a >90% reduction in abundance of viral mRNA reads, while at the same time no such reduction was detected for the abundance of cellular transcripts. The presented data reveal that ribavirin significantly increases the frequency of HRSV-specific RNA mutations, suggesting a direct influence on the fidelity of the HRSV polymerase. The presented data show that transitions and transversions occur during HRSV replication and that these changes occur in hot spots along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, our data indicate that in the continuous cell types used and at the time points analyzed, the abundances of some HRSV mRNAs do not reflect the order in which the mRNAs are transcribed., Importance: Human respiratory syncytial virus (HRSV) is a major pediatric pathogen. Ribavirin can be used in children who are extremely ill to reduce the amount of virus and to lower the burden of disease. Ribavirin is used as an experimental therapy with other viruses. The mechanism of action of ribavirin against HRSV is not well understood, although it is thought to increase the mutation rate of the viral polymerase during replication. To investigate this hypothesis, we used a high-resolution approach that allowed us to determine the genetic sequence of the virus to a great depth of coverage. We found that ribavirin did not cause a detectable change in the relative amounts of viral mRNA transcripts. However, we found that ribavirin treatment did indeed cause an increase in the number of mutations, which was associated with a decrease in virus production., (Copyright © 2016 Aljabr et al.)

Background: In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated., Results: We assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases., Conclusion: We have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.

Advances in genomic and transcriptome sequencing are revealing the massive scale of previously unrecognised alterations occurring during neoplastic transformation. Breast cancers are genetically and phenotypically heterogeneous. Each of the three major subtypes [ERBB2 amplified, estrogen receptor (ESR)-positive and triple-negative] poses diagnostic and therapeutic challenges. Here we show, using high-resolution next-generation transcriptome sequencing, that in all three breast cancer subtypes, but not matched controls, there was significant overexpression of transcripts from intronic and untranslated regions in addition to exons from specific genes, particularly amplified oncogenes and hormone receptors. For key genes ERBB2 and ESR1, we demonstrate that overexpression is linked to the production of highly modified and truncated splice variants in tumours, but not controls, correlated with tumour subtype. Translation of these tumour-specific splice variants generates truncated proteins with altered subcellular locations and functions, modifying the phenotype, affecting tumour biology, and targeted antitumour therapies. In contrast, tumour suppressors TP53, BRCA1/2 and NF1 did not show intronic overexpression or truncated splice variants in cancers. These findings emphasize the detection of intronic as well as exonic changes in the transcriptional landscapes of cancers have profound therapeutic implications.

Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli., (Copyright © 2015, Veses-Garcia et al.)

Background: Almost all genome sequencing projects neglect the fact that diploid organisms contain two genome copies and consequently what is published is a composite of the two. This means that the relationship between alternate alleles at two or more linked loci is lost. We have developed a simplified method of directly obtaining the haploid sequences of each genome copy from an individual organism., Results: The diploid sequences of three groups of cattle samples were obtained using a simple sample preparation procedure requiring only a microscope and a haemocytometer. Samples were: 1) lymphocytes from a single Angus steer; 2) sperm cells from an Angus bull; 3) lymphocytes from East African Zebu (EAZ) cattle collected and processed in a field laboratory in Eastern Kenya. Haploid sequence from a fosmid library prepared from lymphocytes of an EAZ cow was used for comparison. Cells were serially diluted to a concentration of one cell per microlitre by counting with a haemocytometer at each dilution. One microlitre samples, each potentially containing a single cell, were lysed and divided into six aliquots (except for the sperm samples which were not divided into aliquots). Each aliquot was amplified with phi29 polymerase and sequenced. Contigs were obtained by mapping to the bovine UMD3.1 reference genome assembly and scaffolds were assembled by joining adjacent contigs that were within a threshold distance of each other. Scaffolds that appeared to contain artefacts of CNV or repeats were filtered out leaving scaffolds with an N50 length of 27-133 kb and a 88-98 % genome coverage. SNP haplotypes were assembled with the Single Individual Haplotyper program to generate an N50 size of 97-201 kb but only ~27-68 % genome coverage. This method can be used in any laboratory with no special equipment at only slightly higher costs than conventional diploid genome sequencing. A substantial body of software for analysis and workflow management was written and is available as supplementary data., Conclusions: We have developed a set of laboratory protocols and software tools that will enable any laboratory to obtain haplotype sequences at only modestly greater cost than traditional mixed diploid sequences.

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.

This work presents the Australian Clinical Dosimetry Service's (ACDS) findings of an investigation of systematic discrepancies between treatment planning system (TPS) calculated and measured audit doses. Specifically, a comparison between the Anisotropic Analytic Algorithm (AAA) and other common dose-calculation algorithms in regions downstream (≥2cm) from low-density material in anthropomorphic and slab phantom geometries is presented. Two measurement setups involving rectilinear slab-phantoms (ACDS Level II audit) and anthropomorphic geometries (ACDS Level III audit) were used in conjunction with ion chamber (planar 2D array and Farmer-type) measurements. Measured doses were compared to calculated doses for a variety of cases, with and without the presence of inhomogeneities and beam-modifiers in 71 audits. Results demonstrate a systematic AAA underdose with an average discrepancy of 2.9 ± 1.2% when the AAA algorithm is implemented in regions distal from lung-tissue interfaces, when lateral beams are used with anthropomorphic phantoms. This systemic discrepancy was found for all Level III audits of facilities using the AAA algorithm. This discrepancy is not seen when identical measurements are compared for other common dose-calculation algorithms (average discrepancy -0.4 ± 1.7%), including the Acuros XB algorithm also available with the Eclipse TPS. For slab phantom geometries (Level II audits), with similar measurement points downstream from inhomogeneities this discrepancy is also not seen., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

The Australian Clinical Dosimetry Service (ACDS) has demonstrated the capacity to perform a basic dosimetry audit on all radiotherapy clinics across Australia. During the ACDS's three and a half year trial the majority of the audits were performed using optically stimulated luminescence dosimeters (OSLD) mailed to facilities for exposure to a reference dose, and then returned to the ACDS for analysis. This technical note investigates the stability of the readout process under the large workload of the national dosimetry audit. The OSLD readout uncertainty contributes to the uncertainty of several terms of the dose calculation equation and is a major source of uncertainty in the audit. The standard deviation of four OSLD readouts was initially established at 0.6 %. Measurements over 13 audit batches--each batch containing 200-400 OSLDs--showed variability (0.5-0.9 %) in the readout standard deviation. These shifts have not yet necessitated a change to the audit scoring levels. However, a standard deviation in OSLD readouts greater than 0.9 % will change the audit scoring levels. We identified mechanical wear on the OSLD readout adapter as a cause of variability in readout uncertainty, however, we cannot rule out other causes. Additionally we observed large fluctuations in the distribution of element correction factors (ECF) for OSLD batches. We conclude that the variability in the width of the ECF distribution from one batch to another is not caused by variability in readout uncertainty, but rather by variations in the OSLD stock.

The evolution of diversity in the marine ecosystem is poorly understood, given the relatively high potential for connectivity, especially for highly mobile species such as whales and dolphins. The killer whale (Orcinus orca) has a worldwide distribution, and individual social groups travel over a wide geographic range. Even so, regional populations have been shown to be genetically differentiated, including among different foraging specialists (ecotypes) in sympatry. Given the strong matrifocal social structure of this species together with strong resource specializations, understanding the process of differentiation will require an understanding of the relative importance of both genetic drift and local adaptation. Here we provide a high-resolution analysis based on nuclear single-nucleotide polymorphic markers and inference about differentiation at both neutral loci and those potentially under selection. We find that all population comparisons, within or among foraging ecotypes, show significant differentiation, including populations in parapatry and sympatry. Loci putatively under selection show a different pattern of structure compared to neutral loci and are associated with gene ontology terms reflecting physiologically relevant functions (e.g. related to digestion). The pattern of differentiation for one ecotype in the North Pacific suggests local adaptation and shows some fixed differences among sympatric ecotypes. We suggest that differential habitat use and resource specializations have promoted sufficient isolation to allow differential evolution at neutral and functional loci, but that the process is recent and dependent on both selection and drift., (© 2014 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Purpose: The Australian Clinical Dosimetry Service (ACDS) has implemented a new method of a nonreference condition Level II type dosimetric audit of radiotherapy services to increase measurement accuracy and patient safety within Australia. The aim of this work is to describe the methodology, tolerances, and outcomes from the new audit., Methods: The ACDS Level II audit measures the dose delivered in 2D planes using an ionization chamber based array positioned at multiple depths. Measurements are made in rectilinear homogeneous and inhomogeneous phantoms composed of slabs of solid water and lung. Computer generated computed tomography data sets of the rectilinear phantoms are supplied to the facility prior to audit for planning of a range of cases including reference fields, asymmetric fields, and wedged fields. The audit assesses 3D planning with 6 MV photons with a static (zero degree) gantry. Scoring is performed using local dose differences between the planned and measured dose within 80% of the field width. The overall audit result is determined by the maximum dose difference over all scoring points, cases, and planes. Pass (Optimal Level) is defined as maximum dose difference ≤3.3%, Pass (Action Level) is ≤5.0%, and Fail (Out of Tolerance) is >5.0%., Results: At close of 2013, the ACDS had performed 24 Level II audits. 63% of the audits passed, 33% failed, and the remaining audit was not assessable. Of the 15 audits that passed, 3 were at Pass (Action Level). The high fail rate is largely due to a systemic issue with modeling asymmetric 60° wedges which caused a delivered overdose of 5%-8%., Conclusions: The ACDS has implemented a nonreference condition Level II type audit, based on ion chamber 2D array measurements in an inhomogeneous slab phantom. The powerful diagnostic ability of this audit has allowed the ACDS to rigorously test the treatment planning systems implemented in Australian radiotherapy facilities. Recommendations from audits have led to facilities modifying clinical practice and changing planning protocols.

The identification of nucleic acid aptamers would be advanced if they could be obtained after fewer rounds of selection and amplification. In this paper the identification of bivalent aptamers for thrombin by SELEX and single-step selection are compared using next generation sequencing and motif finding informatics. Results show that similar aptamers are identified by both methods. This is significant because it shows that next generation sequencing and motif finding informatics have the potential to simplify the selection of aptamers by avoiding multiple rounds of enzymatic transcription and amplification.

Purpose: The purpose of this investigation was to assess the angular dependence of a commercial optically stimulated luminescence dosimeter (OSLD) dosimetry system in MV x-ray beams at depths beyond d(max) and to find ways to mitigate this dependence for measurements in phantoms., Methods: Two special holders were designed which allow a dosimeter to be rotated around the center of its sensitive volume. The dosimeter's sensitive volume is a disk, 5 mm in diameter and 0.2 mm thick. The first holder rotates the disk in the traditional way. It positions the disk perpendicular to the beam (gantry pointing to the floor) in the initial position (0°). When the holder is rotated the angle of the disk towards the beam increases until the disk is parallel with the beam ("edge on," 90°). This is referred to as Setup 1. The second holder offers a new, alternative measurement position. It positions the disk parallel to the beam for all angles while rotating around its center (Setup 2). Measurements with five to ten dosimeters per point were carried out for 6 MV at 3 and 10 cm depth. Monte Carlo simulations using GEANT4 were performed to simulate the response of the active detector material for several angles. Detector and housing were simulated in detail based on microCT data and communications with the manufacturer. Various material compositions and an all-water geometry were considered., Results: For the traditional Setup 1 the response of the OSLD dropped on average by 1.4% ± 0.7% (measurement) and 2.1% ± 0.3% (Monte Carlo simulation) for the 90° orientation compared to 0°. Monte Carlo simulations also showed a strong dependence of the effect on the composition of the sensitive layer. Assuming the layer to completely consist of the active material (Al2O3) results in a 7% drop in response for 90° compared to 0°. Assuming the layer to be completely water, results in a flat response within the simulation uncertainty of about 1%. For the new Setup 2, measurements and Monte Carlo simulations found the angular dependence of the dosimeter to be below 1% and within the measurement uncertainty., Conclusions: The dosimeter system exhibits a small angular dependence of approximately 2% which needs to be considered for measurements involving other than normal incident beams angles. This applies in particular to clinical in vivo measurements where the orientation of the dosimeter is dictated by clinical circumstances and cannot be optimized as otherwise suggested here. When measuring in a phantom, the proposed new setup should be considered. It changes the orientation of the dosimeter so that a coplanar beam arrangement always hits the disk shaped detector material from the thin side and thereby reduces the angular dependence of the response to within the measurement uncertainty of about 1%. This improvement makes the dosimeter more attractive for clinical measurements with multiple coplanar beams in phantoms, as the overall measurement uncertainty is reduced. Similarly, phantom based postal audits can transition from the traditional TLD to the more accurate and convenient OSLD.