Validation and development of sequence-based tools to analyse the human gut virome

The gut microbiome is a complex community of microorganisms that interacts closely with the human host and is believed to play an important role in the maintenance of human health. The viral component of this community is referred to as the human gut virome and is dominated by bacteriophage. Bacteriophage are central to microbial ecosystems by facilitating nutrient turnover, horizontal gene transfer and driving bacterial diversity. In this way the gut virome is believed to closely interact with the human host by shaping the composition and function of the gut microbiome. However, the gut virome also represents one of the biggest gaps in our understanding of the microbiome as it is dominated by unknown bacteriophage targeting unknown bacterial hosts and with uncharacterised downstream functions. These challenges mean that virome research relies heavily on sequence-based approaches and metagenomics to identify compositional patterns and targets for future characterisation. A typical virome study involves physical and chemical separation of individual virions from the cellular components of the microbiome and the contents of the faecal, luminal or mucosal sample from which it came. A viral metagenome is then generated by extracting virome DNA and/or RNA for sequencing on a given platform. These sequencing reads are then quality filtered and assembled to reconstruct the viral genomes in the original sample. The abundance of these assemblies is then estimated by aligning the sequencing reads and performing statistical analysis. However, each step in a virome analysis pipeline has the potential to distort the final viral community and given the unknown nature of the virome, this distortion is difficult to identify and characterise. As a result, conclusions are often drawn from virome studies without fully appreciating the impact of the analysis methods on the findings. This thesis examines the major steps in sequence-based virome analysis pipelines, highlighting how choices made at each step of an analysis protocol can impact the final conclusions drawn from a study. In doing so, we have changed our perspective of the human gut virome and challenged previous assumptions. Chapter One discusses the current understanding of the virome field, giving particular attention to how the analysis methods and challenges affect our view of the virome. In Chapter Two, we focus on the assembly step of virome analysis pipelines. This step is of particular importance to virome studies, as an assembler’s ability to recover viral sequences can ultimately determine the amount of sequence information used in a that study. We compared all short-read assembly programs used in virome studies to date, across mock communities, simulated and real datasets. We found that not all assemblers are equal, and choice of assembler can drastically affect the conclusions that can be drawn from a virome study. These findings call the comparability of different virome studies into question and would suggest that previous virome studies would benefit from reanalysis using improved assembly methods and re-examination of the conclusions drawn. As discussed, the human gut virome is dominated by “viral dark matter”; those sequences which do not share homology to reference databases. However, the majority of what is currently known about the virome in human health and disease is based on the minor fraction of viral sequences collated in these databases. This presents a serious gap in our understanding and was the primary focus of Chapter Three. We reanalysed a keystone inflammatory bowel disease (IBD) dataset, which had formed the foundation of much of what we knew about the virome in IBD. We developed a new approach to analysing the virome beyond the identifiable minority and by doing so, changed our understanding of the virome in IBD significantly. In the final chapter, we directed our attention to possibly the most important aspect of a sequence-based study, the sequencing approach itself. This step bridges the gap between the biological information in a virome and the digital information that is analysed. As with all steps in a virome analysis pipeline, this has serious implications for the final conclusions of the study. We described the use of long-read sequencing in the human gut virome and the benefits and challenges which are associated with this technology. We also found the ability of amplified short-read sequencing libraries to represent the gut virome was limited, but that alternative library preparation methods and long-read sequencing platforms may be able to address these limitations. These findings imply that much of what we know about that human gut virome may be linked to sequencing performance, rather than the biology of the community itself. These three major aspects of virome analysis pipelines highlight the importance of considering the impact of the analysis approach when interpreting the results of virome data and complex biological systems in general

Gut bacteriophage: Current understanding and challenges

The gut microbiome is widely accepted to have a significant impact on human health yet, despite years of research on this complex ecosystem, the contributions of different forces driving microbial population structure remain to be fully elucidated. The viral component of the human gut microbiome is dominated by bacteriophage, which are known to play crucial roles in shaping microbial composition, driving bacterial diversity, and facilitating horizontal gene transfer. Bacteriophage are also one of the most poorly understood components of the human gut microbiome, with the vast majority of viral sequences sharing little to no homology to reference databases. If we are to understand the dynamics of bacteriophage populations, their interaction with the human microbiome and ultimately their influence on human health, we will depend heavily on sequence based approaches and in silico tools. This is complicated by the fact that, as with any research field in its infancy, methods of analyses vary and this can impede our ability to compare the outputs of different studies. Here, we discuss the major findings to date regarding the human virome and reflect on our current understanding of how gut bacteriophage shape the microbiome. We consider whether or not the virome field is built on unstable foundations and if so, how can we provide a solid basis for future experimentation. The virome is a challenging yet crucial piece of the human microbiome puzzle. In order to develop our understanding, we will discuss the need to underpin future studies with robust research methods and suggest some solutions to existing challenges

Choice of assembly software has a critical impact on virome characterisation

peer-reviewedBackground The viral component of microbial communities plays a vital role in driving bacterial diversity, facilitating nutrient turnover and shaping community composition. Despite their importance, the vast majority of viral sequences are poorly annotated and share little or no homology to reference databases. As a result, investigation of the viral metagenome (virome) relies heavily on de novo assembly of short sequencing reads to recover compositional and functional information. Metagenomic assembly is particularly challenging for virome data, often resulting in fragmented assemblies and poor recovery of viral community members. Despite the essential role of assembly in virome analysis and difficulties posed by these data, current assembly comparisons have been limited to subsections of virome studies or bacterial datasets. Design This study presents the most comprehensive virome assembly comparison to date, featuring 16 metagenomic assembly approaches which have featured in human virome studies. Assemblers were assessed using four independent virome datasets, namely, simulated reads, two mock communities, viromes spiked with a known phage and human gut viromes. Results Assembly performance varied significantly across all test datasets, with SPAdes (meta) performing consistently well. Performance of MIRA and VICUNA varied, highlighting the importance of using a range of datasets when comparing assembly programs. It was also found that while some assemblers addressed the challenges of virome data better than others, all assemblers had limitations. Low read coverage and genomic repeats resulted in assemblies with poor genome recovery, high degrees of fragmentation and low-accuracy contigs across all assemblers. These limitations must be considered when setting thresholds for downstream analysis and when drawing conclusions from virome data

Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa

peer-reviewedBackground Faecalibacterium prausnitzii is a ubiquitous member of the human gut microbiome, constituting up to 15% of the total bacteria in the human gut. Substantial evidence connects decreased levels of F. prausnitzii with the onset and progression of certain forms of inflammatory bowel disease, which has been attributed to its anti-inflammatory potential. Two phylogroups of F. prausnitzii have been identified, with a decrease in phylogroup I being a more sensitive marker of intestinal inflammation. Much of the genomic and physiological data available to date was collected using phylogroup II strains. Little analysis of F. prausnitzii genomes has been performed so far and genetic differences between phylogroups I and II are poorly understood. Results In this study we sequenced 11 additional F. prausnitzii genomes and performed comparative genomics to investigate intraspecies diversity, functional gene complement and the mobilome of 31 high-quality draft and complete genomes. We reveal a very low level of average nucleotide identity among F. prausnitzii genomes and a high level of genome plasticity. Two genomogroups can be separated based on differences in functional gene complement, albeit that this division does not fully agree with separation based on conserved gene phylogeny, highlighting the importance of horizontal gene transfer in shaping F. prausnitzii genomes. The difference between the two genomogroups is mainly in the complement of genes associated with catabolism of carbohydrates (such as a predicted sialidase gene in genomogroup I) and amino acids, as well as defense mechanisms. Conclusions Based on the combination of ANI of genomic sequences, phylogenetic analysis of core proteomes and functional differences we propose to separate the species F. prausnitzii into two new species level taxa: F. prausnitzii sensu stricto (neotype strain A2–165T = DSM 17677T = JCM 31915T) and F. moorei sp. nov. (type strain ATCC 27768T = NCIMB 13872T).This research was conducted with the financial support of Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273, a Science Foundation Ireland’s Spokes Programme which is co-funded under the European Regional Development Fund under Grant Number SFI/14/SP APC/B3032, and a research grant from Janssen Biotech, Inc

Anatomy of a cluster IDP. Part 2: Noble gas abundances, trace element geochemistry, isotopic abundances, and trace organic chemistry of several fragments from L2008#5

The topics discussed include the following: noble gas content and release temperatures; trace element abundances; heating summary of cluster fragments; isotopic measurements; and trace organic chemistry

Dynamics of Light Antiquarks in the Proton

We present a comprehensive analysis of the recent data from the E866 experiment at Fermilab on Drell-Yan production in pD and pp collisions, which indicates a non-trivial x-dependence for the asymmetry between u-bar and d-bar quark distributions in the proton. The relatively fast decrease of the asymmetry at large x suggests the important role played by the chiral structure of the nucleon, in particular the pi-N and pi-Delta components of the nucleon wave function. At small x the data require an additional non-chiral component, which may be attributed to the Pauli exclusion principle as first suggested by Field and Feynman.Comment: version to appear in Phys. Rev.

How old is the Tasmanian cultural landscape? a test of landscape openness using quantitative land-cover reconstructions

Aim: To test competing hypotheses about the timing and extent of Holocene landscape opening using pollen-based quantitative land-cover estimates. Location: Dove Lake, Tasmanian Wilderness World Heritage Area, Australia. Methods: Fossil pollen data were incorporated into pollen dispersal models and corrected for differences in pollen productivity among key plant taxa. Mechanistic models (REVEALS-Regional Estimates of VEgetation Abundance from Large Sites) employing different models for pollen dispersal (Gaussian plume and Lagrangian stochastic models) were evaluated and applied in the Southern Hemisphere for the first time. Results: Validation of the REVEALS model with vegetation cover data suggests an overall better performance of the Lagrangian stochastic model. Regional land-cover estimates for forest and non-forest plant taxa show persistent landscape openness throughout the Holocene (average landscape openness similar to 50%). Gymnoschoenus sphaerocephalus, an indicator of moorland vegetation, shows higher values during the early Holocene (11.7-9 ka) and declines slightly through the mid-Holocene (9-4.5 ka) during a phase of partial landscape afforestation. Rain forest cover reduced (from similar to 40% to similar to 20%) during the period between 4.2-3.5 ka. Main conclusions: Pollen percentages severely under-represent landscape openness in western Tasmania and this bias has fostered an over-estimation of Holocene forest cover from pollen data. Treeless vegetation dominated Holocene landscapes of the Dove Lake area, allowing us to reject models of landscape evolution that invoke late-Holocene replacement of a rain forest-dominated landscape by moorland. Instead, we confirm a model of Late Pleistocene inheritance of open vegetation. Rapid forest decline occurred after c.4 ka, likely in response to regional moisture decline.Australian Research Council; AINSE AWARD [ALNGRA16024]; AINSE PGRA scholarship [12039]info:eu-repo/semantics/publishedVersio

dbar/ubar Asymmetry and the Origin of the Nucleon Sea

The Drell-Yan cross section ratios, $\sigma(p+d)/\sigma(p+p)$, measured in Fermilab E866, have led to the first determination of $\bar d(x) / \bar u(x)$, $\bar d(x) - \bar u(x)$, and the integral of $\bar d(x) - \bar u(x)$ for the proton over the range $0.02 \le x \le 0.345$. The E866 results are compared with predictions based on parton distribution functions and various theoretical models. The relationship between the E866 results and the NMC measurement of the Gottfried integral is discussed. The agreement between the E866 results and models employing virtual mesons indicates these non-perturbative processes play an important role in the origin of the $\bar d$, $\bar u$ asymmetry in the nucleon sea.Comment: 5 pages, 3 figures, ReVTe

Feeding the rural tourism strategy? Food and notions of place and identity

The humble rural cuisine has now been thrust at the forefront of economic development strategies. This conceptual paper is a contribution to a growing critical awareness of the operations of the food industry and helps to foster a critical understanding of how, if at all, local food and its associated culture can help sustain rural tourism particularly and rural communities generally. It is inspired by literature about the international political economy of food and the many experiences of local food development, and is aware of the contrast between the structure of the industry and the hopes associated with its practice on the ground. The paper thus argues that, beyond the glamour and hype, there are those who gain, as well as those who lose, from the current food fad. While it explains the causes of the contemporary craze with food, the paper also interrogates the naı¨ve expectations often placed in food as a motor of rural development, and as the panacea for struggling rural communities. The empirical data on which this chapter is based are drawn from 18 short chapters explaining the history of various “traditional dishes” from the islands of the broad North Atlantic that feature in a recent food publication.peer-reviewe

Perovskite-perovskite tandem photovoltaics with optimized bandgaps

We demonstrate four and two-terminal perovskite-perovskite tandem solar cells with ideally matched bandgaps. We develop an infrared absorbing 1.2eV bandgap perovskite, $FA_{0.75}Cs_{0.25}Sn_{0.5}Pb_{0.5}I_3$, that can deliver 14.8 % efficiency. By combining this material with a wider bandgap $FA_{0.83}Cs_{0.17}Pb(I_{0.5}Br_{0.5})_3$ material, we reach monolithic two terminal tandem efficiencies of 17.0 % with over 1.65 volts open-circuit voltage. We also make mechanically stacked four terminal tandem cells and obtain 20.3 % efficiency. Crucially, we find that our infrared absorbing perovskite cells exhibit excellent thermal and atmospheric stability, unprecedented for Sn based perovskites. This device architecture and materials set will enable 'all perovskite' thin film solar cells to reach the highest efficiencies in the long term at the lowest costs