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Sequence variation observed in populations of pathogens can be used for important public health and evolutionary genomic analyses, especially outbreak analysis and transmission reconstruction. Identifying this variation is typically achieved by aligning sequence reads to a reference genome, but this approach is susceptible to reference biases and requires careful filtering of called genotypes. There is a need for tools that can process this growing volume of bacterial genome data, providing rapid results, but that remain simple so they can be used without highly trained bioinformaticians, expensive data analysis, and long-term storage and processing of large files. Here we describe split k-mer analysis (SKA2), a method that supports both reference-free and reference-based mapping to quickly and accurately genotype populations of bacteria using sequencing reads or genome assemblies. SKA2 is highly accurate for closely related samples, and in outbreak simulations, we show superior variant recall compared with reference-based methods, with no false positives. SKA2 can also accurately map variants to a reference and be used with recombination detection methods to rapidly reconstruct vertical evolutionary history. SKA2 is many times faster than comparable methods and can be used to add new genomes to an existing call set, allowing sequential use without the need to reanalyze entire collections. With an inherent absence of reference bias, high accuracy, and a robust implementation, SKA2 has the potential to become the tool of choice for genotyping bacteria. SKA2 is implemented in Rust and is freely available as open-source software.

Studies of bacterial adaptation and evolution are hampered by the difficulty of measuring traits such as virulence, drug resistance, and transmissibility in large populations. In contrast, it is now feasible to obtain high-quality complete assemblies of many bacterial genomes thanks to scalable high-accuracy long-read sequencing technologies. To exploit this opportunity, we introduce a phenotype- and alignment-free method for discovering coselected and epistatically interacting genomic variation from genome assemblies covering both core and accessory parts of genomes. Our approach uses a compact colored de Bruijn graph to approximate the intragenome distances between pairs of loci for a collection of bacterial genomes to account for the impacts of linkage disequilibrium (LD). We demonstrate the versatility of our approach to efficiently identify associations between loci linked with drug resistance and adaptation to the hospital niche in the major human bacterial pathogens Streptococcus pneumoniaeand Enterococcus faecalis.

Bacterial genomes differ in both gene content and sequence mutations, which underlie extensive phenotypic diversity, including variation in susceptibility to antimicrobials or vaccine-induced immunity. To identify and quantify important variants, all genes within a population must be predicted, functionally annotated, and clustered, representing the “pangenome.” Despite the volume of genome data available, gene prediction and annotation are currently conducted in isolation on individual genomes, which is computationally inefficient and frequently inconsistent across genomes. Here, we introduce the open-source software graph-gene-caller (ggCaller). ggCaller combines gene prediction, functional annotation, and clustering into a single workflow using population-wide de Bruijn graphs, removing redundancy in gene annotation and resulting in more accurate gene predictions and orthologue clustering. We applied ggCaller to simulated and real-world bacterial data sets containing hundreds or thousands of genomes, comparing it to current state-of-the-art tools. ggCaller has considerable speed-ups with equivalent or greater accuracy, particularly with data sets containing complex sources of error, such as assembly contamination or fragmentation. ggCaller is also an important extension to bacterial genome-wide association studies, enabling querying of annotated graphs for functional analyses. We highlight this application by functionally annotating DNA sequences with significant associations to tetracycline and macrolide resistance in Streptococcus pneumoniae, identifying key resistance determinants that were missed when using only a single reference genome. ggCaller is a novel bacterial genome analysis tool with applications in bacterial evolution and epidemiology.

Bacterial genome data are accumulating at an unprecedented speed due to the routine use of sequencing in clinical diagnoses, public health surveillance, and population genetics studies. Genealogical reconstruction is fundamental to many of these uses; however, inferring genealogy from large-scale genome data sets quickly, accurately, and flexibly is still a challenge. Here, we extend an alignment- and annotation-free method, PopPUNK, to increase its flexibility and interpretability across data sets. Our method, iterative-PopPUNK, rapidly produces multiple consistent cluster assignments across a range of sequence identities. By constructing a partially resolved genealogical tree with respect to these clusters, users can select a resolution most appropriate for their needs. We showed the accuracy of clusters at all levels of similarity and genealogical inference of iterative-PopPUNK based on simulated data and obtained phylogenetically concordant results in real data sets from seven bacterial species. Using two example sets of Escherichia/Shigellaand Vibrio parahaemolyticusgenomes, we show that iterative-PopPUNK can achieve cluster resolutions ranging from phylogroup down to sequence typing (ST). The iterative-PopPUNK algorithm is implemented in the “PopPUNK_iterate” program, available as part of the PopPUNK package.

England's COVID-19 roadmap out of lockdown policy set out the timeline and conditions for the stepwise lifting of non-pharmaceutical interventions (NPIs) as vaccination roll-out continued, with step one starting on March 8, 2021. In this study, we assess the roadmap, the impact of the delta (B.1.617.2) variant of SARS-CoV-2, and potential future epidemic trajectories.

Streptococcus pneumoniaecan be divided into many strains, each a distinct set of isolates sharing similar core and accessory genomes, which co-circulate within the same hosts. Previous analyses suggested the short-term vaccine-associated dynamics of S. pneumoniaestrains may be mediated through multi-locus negative frequency-dependent selection (NFDS), which maintains accessory loci at equilibrium frequencies. Long-term simulations demonstrated NFDS stabilised clonally-evolving multi-strain populations through preventing the loss of variation through drift, based on polymorphism frequencies, pairwise genetic distances and phylogenies. However, allowing symmetrical recombination between isolates evolving under multi-locus NFDS generated unstructured populations of diverse genotypes. Replication of the observed data improved when multi-locus NFDS was combined with recombination that was instead asymmetrical, favouring deletion of accessory loci over insertion. This combination separated populations into strains through outbreeding depression, resulting from recombinants with reduced accessory genomes having lower fitness than their parental genotypes. Although simplistic modelling of recombination likely limited these simulations’ ability to maintain some properties of genomic data as accurately as those lacking recombination, the combination of asymmetrical recombination and multi-locus NFDS could restore multi-strain population structures from randomised initial populations. As many bacteria inhibit insertions into their chromosomes, this combination may commonly underlie the co-existence of strains within a niche.

Streptococcus pneumoniaecan be divided into many strains, each a distinct set of isolates sharing similar core and accessory genomes, which co-circulate within the same hosts. Previous analyses suggested the short-term vaccine-associated dynamics of S. pneumoniaestrains may be mediated through multi-locus negative frequency-dependent selection (NFDS), which maintains accessory loci at equilibrium frequencies. Long-term simulations demonstrated NFDS stabilised clonally-evolving multi-strain populations through preventing the loss of variation through drift, based on polymorphism frequencies, pairwise genetic distances and phylogenies. However, allowing symmetrical recombination between isolates evolving under multi-locus NFDS generated unstructured populations of diverse genotypes. Replication of the observed data improved when multi-locus NFDS was combined with recombination that was instead asymmetrical, favouring deletion of accessory loci over insertion. This combination separated populations into strains through outbreeding depression, resulting from recombinants with reduced accessory genomes having lower fitness than their parental genotypes. Although simplistic modelling of recombination likely limited these simulations’ ability to maintain some properties of genomic data as accurately as those lacking recombination, the combination of asymmetrical recombination and multi-locus NFDS could restore multi-strain population structures from randomised initial populations. As many bacteria inhibit insertions into their chromosomes, this combination may commonly underlie the co-existence of strains within a niche.

The burden of malaria is heavily concentrated in sub-Saharan Africa (SSA) where cases and deaths associated with COVID-19 are rising1. In response, countries are implementing societal measures aimed at curtailing transmission of SARS-CoV-22,3. Despite these measures, the COVID-19 epidemic could still result in millions of deaths as local health facilities become overwhelmed4. Advances in malaria control this century have been largely due to distribution of long-lasting insecticidal nets (LLINs)5, with many SSA countries having planned campaigns for 2020. In the present study, we use COVID-19 and malaria transmission models to estimate the impact of disruption of malaria prevention activities and other core health services under four different COVID-19 epidemic scenarios. If activities are halted, the malaria burden in 2020 could be more than double that of 2019. In Nigeria alone, reducing case management for 6 months and delaying LLIN campaigns could result in 81,000 (44,000–119,000) additional deaths. Mitigating these negative impacts is achievable, and LLIN distributions in particular should be prioritized alongside access to antimalarial treatments to prevent substantial malaria epidemics.

Group A Streptococcus(GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies.

The routine use of genomics for disease surveillance provides the opportunity for high-resolution bacterial epidemiology. Current whole-genome clustering and multilocus typing approaches do not fully exploit core and accessory genomic variation, and they cannot both automatically identify, and subsequently expand, clusters of significantly similar isolates in large data sets spanning entire species. Here, we describe PopPUNK (Population Partitioning Using Nucleotide K-mers), a software implementing scalable and expandable annotation- and alignment-free methods for population analysis and clustering. Variable-length k-mer comparisons are used to distinguish isolates’ divergence in shared sequence and gene content, which we demonstrate to be accurate over multiple orders of magnitude using data from both simulations and genomic collections representing 10 taxonomically widespread species. Connections between closely related isolates of the same strain are robustly identified, despite interspecies variation in the pairwise distance distributions that reflects species’ diverse evolutionary patterns. PopPUNK can process 103–104genomes in a single batch, with minimal memory use and runtimes up to 200-fold faster than existing model-based methods. Clusters of strains remain consistent as new batches of genomes are added, which is achieved without needing to reanalyze all genomes de novo. This facilitates real-time surveillance with consistent cluster naming between studies and allows for outbreak detection using hundreds of genomes in minutes. Interactive visualization and online publication is streamlined through the automatic output of results to multiple platforms. PopPUNK has been designed as a flexible platform that addresses important issues with currently used whole-genome clustering and typing methods, and has potential uses across bacterial genetics and public health research.

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ABSTRACTThis article looks at work conditions in our current age brought about by materialism. In particular, it looks at the damaging effects of materialistic scientific management principles and the allied condition of illusionism. It shows how the policies of society based on neoliberal managed care principles are not only inadequate for addressing the problem but actually exacerbate it since they adopt the self-same materialistic principles. In contrast, it argues for a psychotherapeutic response based on the principles of anthroposophic psychotherapy and other similarly socially and politically aware therapies such as intersubjectivity and relational psychoanalysis.

Detectors capable of withstanding high radiation environments for prolonged periods of exposure are essential for the monitoring of nuclear power stations and nuclear waste as well as for space exploration. Schottky diode X-ray detectors were exposed to high dose proton irradiation (1013 cm-2, 50 MeV) and changes in the detection resolution (spectroscopic full width half-maximum) have been observed. Using Deep Level Transient Spectroscopy (DLTS) and the degradation of the electrical characteristics of the diode, we have shown that radiation induced traps located in the upper half of the bandgap have reduced the concentration of carriers.

A report on the seventh annual Infectious Disease Genomics Conference, held in Hinxton, Cambridge, UK, 14–16 October 2015.

This article investigates the experience of loss and bereavement from the point of view of a spiritual perspective on the phenomenon. It also looks at the experience of holding a marginalized viewpoint within an academic and professional community and adopts an autoethnographic approach to the inquiry. Some conclusions are drawn from the study in terms of its implications for theory and professional life.

This paper explores the application of reflexive action research in practice by examining a piece of the author's previously published work. A process of inquiry is described that involves reflexively engaging with original formulations in order to further continuing professional development. Guidelines for conducting this kind of research are described.

This paper gives an overview of different ways of conducting counselling and psychotherapy research. An approach to research is described which overcomes the research-practice gap: reflexive action research. It is argued that this approach to research is particularly appropriate and relevant for practitioners since it draws upon skills and awareness necessary for clinical work, and can complement positivist methods of research which are already well established in the profession. The wider use of systematic clinical case studies is suggested as a means of promoting the reflexive action research approach. Further consideration of issues of validity and reliability is required on the part of those intending to apply this approach within the field of therapy research.

A SARS-CoV-2 transmission model fitted to surveillance data estimates virus transmissibility, infection severity, and the impact of interventions.

IntroduzioneÈ apparsa evidente, sin dall'inizio, la necessità di una ricerca di équipe per affrontare il tema delle commissioni delle assemblee legislative in una prospettiva comparata piú ampia di quelle sin qui condotte. A tal fine si è deciso di affidare la ricerca ad uno studioso per ciascuno dei Paesi prescelti, senza predeterminazione di modelli teorici rigidi, sia perché si era constatata una notevole carenza di dati di fatto in riferimento a numerosi Paesi, sia perché vi era la convinzione che il tradizionale approccio anglo-americano tendeva a sopravvalutare, a fini comparati generali, i risultati delle ricerche condotte in riferimento alle commissioni delle assemblee legislative statunitensi e britanniche.

AbstractA discussion of the various elements which influence counselling in GP surgeries - clients, organizational culture, and the counsellor's professional development. The article will examine these elements in the light of some basic psychodynamic theory. It will argue that there are aspects of working in this setting that are common to all situations, and that there are also aspects which are unique to each setting.

This short paper describes a recent visit to an exhibition of modern art. It examines my impressions in the light of contemporary psychodynamic theory. The exhibition was called 'Some Went Mad, Some Ran Away…' It included the work of fifteen modern artists, mainly created between 1992 and 1994, and selected by the British artist Damien Hirst. The exhibition leaflet said that the artists had used 'real objects, body-casts, natural and man-made forms, sculpture, painting and photography, to set up a number of provocative juxtapositions'.

In this article I will adopt the viewpoint that current social issues, concerns and preoccupations are mirrored to some extent in films, and indeed culture in general - a view which has been expressed by many people in one form or another (see, for example, Caldwell [1996] or Sekoff [1989: 148] who says that 'film must be understood as potentially embodying, disguising, challenging, containing, exploring, producing and so on the conditions of our cultural and personal lives, as well as the meanings we weave around them'). For example, the depression, unemployment and political polarization of the 1920s and 1930s produced two types of film. There were those which directly mirrored the political developments and social hardship of the times - such as the Charlie Chaplin films - and those which created a fantasy world of make-believe in order to escape the harsh realities of the day - for instance, the Hollywood Busby Berkeley musicals. So what are films telling us about society today?

Population-level comparisons of prokaryotic genomes must take into account the substantial differences in gene content resulting from horizontal gene transfer, gene duplication and gene loss. However, the automated annotation of prokaryotic genomes is imperfect, and errors due to fragmented assemblies, contamination, diverse gene families and mis-assemblies accumulate over the population, leading to profound consequences when analysing the set of all genes found in a species. Here, we introduce Panaroo, a graph-based pangenome clustering tool that is able to account for many of the sources of error introduced during the annotation of prokaryotic genome assemblies. Panaroo is available at https://github.com/gtonkinhill/panaroo.

Being able to identify the genetic variants responsible for specific bacterial phenotypes has been the goal of bacterial genetics since its inception and is fundamental to our current level of understanding of bacteria. This identification has been based primarily on painstaking experimentation, but the availability of large data sets of whole genomes with associated phenotype metadata promises to revolutionize this approach, not least for important clinical phenotypes that are not amenable to laboratory analysis. These models of phenotype-genotype association can in the future be used for rapid prediction of clinically important phenotypes such as antibiotic resistance and virulence by rapid-turnaround or point-of-care tests. However, despite much effort being put into adapting genome-wide association study (GWAS) approaches to cope with bacterium-specific problems, such as strong population structure and horizontal gene exchange, current approaches are not yet optimal. We describe a method that advances methodology for both association and generation of portable prediction models.

PRESSURES in ultra-high pressure apparatus of the belt or anvil1types are commonly inferred from a subsidiary load/pressure calibration using the well-known resistance discontinuities in bismuth at 25.4 kilobars2, in thallium at 36.7 kb2and in barium at 59 kb3. Possible uncertainties of this method are associated with: (a) variations in the anvil4and test-cell materials5; (b) the influence of sample geometry6; (c) the influence of temperature, particularly when generated within the test-cell1; (d) the rather wide region between 0 and 25.4 kb for which no very suitable resistance transition is yet available. In this communication a load discontinuity method is described which extends load/pressure calibration for ultra-high pressure apparatus. It is applied here to a ¾-in. edge tetrahedral unit of the N.B.S. type1.

It is generally believed that pregnancy predisposes to disease of the biliary tract. In previous papers one of us1 was concerned in reports of relief of biliary pain following cholecystectomy. Pain seemed to be due to an increased pressure in the bile ducts resulting from an obstruction at the lower end of the common bile duct due to a muscle spasm. This spasm seemed to be in the muscle in the second portion of the duodenal wall. This spasm and the resulting pressure and pain were relieved by the use of amyl nitrite and glyceryl trinitrate.Recently in a case of intractable vomiting of pregnancy in which jaundice had begun to develop, it seemed possible that such a spasm might be a causative factor. Glyceryl trinitrate was given after each meal with the result that vomiting stopped and a proposed therapeutic abortion was rendered unnecessary. This experience suggested the

An amendment to this paper has been published and can be accessed via a link at the top of the paper.