16S rRNA gene sequencing of mock microbial populations- impact of DNA extraction method, primer choice and sequencing platform

peer-reviewedBackground Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA. Results The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar. Conclusions Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Numbers SFI/12/RC/2273 and 11/PI/1137 and by FP7 funded CFMATTERS (Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified, Grant Agreement no. 603038)

Simultaneous incremental neuroevolution of motor control, navigation and object manipulation in 3D virtual creatures

There have been numerous attempts to develop 3D virtual agents by applying evolutionary processes to populations that exist in a realistic physical simulation. Whilst often contributing useful knowledge, no previous work has demonstrated the capacity to evolve a sequence of increasingly complex behaviours in a single, unified system. This thesis has this demonstration as its primary aim. A rigorous exploration of one aspect of incremental artificial evolution was carried out to understand how subtask presentations affect the whole-task generalisation performance of evolved, fixed-morphology 3D agents. Results from this work led to the design of an environment–body–control architecture that can be used as a base for evolving multiple behaviours incrementally. A simulation based on this architecture with a more complex environment was then developed and explored. This system was then adapted to include elements of physical manipulation as a first step toward a fully physical virtual creature environment demonstrating advanced evolved behaviours. The thesis demonstrates that incremental evolutionary systems can be subject to problems of forgetting and loss of gradient, and that different complexification strategies have a strong bearing on the management of these issues. Presenting successive generations of the population to a full range of objective functions (covering and revisiting the range of complexity) outperforms straightforward linear or direct presentations, establishing a more robust approach to the evolution of naturalistic embodied agents. When combining this approach with a bespoke control architecture in a problem requiring reactive and deliberative behaviours, we see results that not only demonstrate success at the tasks, but also show a variety of intricate behaviours being used. This is the first ever example of the simultaneous incremental evolution in 3D of composite behaviours more complex than simple locomotion. Finally, the architecture demonstrably supports extension to manipulation in a feedback control task. Given the problem-agnostic controller architecture, these results indicate a system with potential for discovering yet more advanced behaviours in yet more complex environments

Lexicase Selection for Multi-Task Evolutionary Robotics

In Evolutionary Robotics, Lexicase selection has proven effective when a single task is broken down into many individual parameterizations. Evolved individuals have generalized across unique configurations of an overarching task. Here, we investigate the ability of Lexicase selection to generalize across multiple tasks, with each task again broken down into many instances. There are three objectives: to determine the feasibility of introducing additional tasks to the existing platform; to investigate any consequential effects of introducing these additional tasks during evolutionary adaptation; and to explore whether the schedule of presentation of the additional tasks over evolutionary time affects the final outcome. To address these aims we use a quadruped animat controlled by a feed-forward neural network with joint-angle, bearing-to-target, and spontaneous sinusoidal inputs. Weights in this network are trained using evolution with Lexicase-based parent selection. Simultaneous adaptation in a wall crossing task (labelled wall-cross) is explored when one of two different alternative tasks is also present: turn-and-seek or cargo-carry. Each task is parameterized into 100 distinct variants, and these variants are used as environments for evaluation and selection with Lexicase. We use performance in a single-task wall-cross environment as a baseline against which to examine the multi-task configurations. In addition, the objective sampling strategy (the manner in which tasks are presented over evolutionary time) is varied, and so data for treatments implementing uniform sampling, even sampling, or degrees of generational sampling are also presented. The Lexicase mechanism successfully integrates evolution of both turn-and-seek and cargo-carry with wall-cross, though there is a performance penalty compared to single task evolution. The size of the penalty depends on the similarity of the tasks. Complementary tasks (wallcross/turn-and-seek) show better performance than antagonistic tasks (wall-cross/cargo-carry). In complementary tasks performance is not affected by the sampling strategy. Where tasks are antagonistic, uniform and even sampling strategies yield significantly better performance than generational sampling. In all cases the generational sampling requires more evaluations and consequently more computational resources. The results indicate that Lexicase is a viable mechanism for multitask evolution of animat neurocontrollers, though the degree of interference between tasks is a key consideration. The results also support the conclusion that the naive, uniform random sampling strategy is the best choice when considering final task performance, simplicity of implementation, and computational efficiency

Objective Sampling Strategies for Generalized Locomotion Behavior with Lexicase Selection

Controllers capable of exhibiting multiple behaviors is a longstanding goal in artificial life. Evolutionary robotics approaches have demonstrated effective optimization of robotic controllers, realizing single behaviors in a variety of domains. However, evolving multiple behaviors in one controller remains an outstanding challenge. Many objective selection algorithms are a potential solution as they are capable of optimizing across tens or hundreds of objectives. In this study, we use Lexicase selection evolving animats capable of both wall crossing and turn/seek behaviors. Our investigation focuses on the objective sampling strategy during selection to balance performance across the two primary tasks. Results show that the sampling strategy does not significantly alter performance, but the number of evaluations required varies significantly across strategies

Incremental Neuroevolution of Reactive and Deliberative 3D Agents

Following earlier work on the neuroevolution of deliberative behaviour to solve increasingly challenging tasks in a twodimensional dynamic world, this paper presents the results of extending the original system to a three-dimensional rigid body simulation. The 3D physically based setting requires that a successful agent continually and deliberately adjust its gait, turning and other motor control over the many stages and sub-stages of these tasks, within its individual evaluation. Achieving such complex interplay between motor control and deliberative control, within a neuroevolutionary framework, is the focus of this work. To this end, a novel neural architecture is presented and an incremental evolutionary approach used to bootstrap the locomotive behaviour of the agents. Agent morphology is fixed as a quadruped with three degrees of freedom per limb. Agent populations have no initial knowledge of the problem domain, and evolve to move around and then solve progressively more difficult challenges in the environment using a tournament-based co-evolutionary algorithm. The results demonstrate not only success at the tasks but also a variety of intricate lifelike behaviours being used, separately and in combination, to achieve this success. Given the problem-agnostic controller architecture, these results indicate a potential for discovering yet more advanced behaviours in yet more complex environments

Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

peer-reviewedRapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number ofmethodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance inmicrobiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced withMiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.This publication has emanated from research supported in part by a research grant from Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273 and 11/PI/1137 and by FP7 funded CFMATTERS (Cystic Fibrosis Microbiome-determined Antibiotic Therapy Trial in Exacerbations: Results Stratified, Grant Agreement no. 603038)

The early decline in renal function in patients with type 1 diabetes and proteinuria predicts the risk of end stage renal disease

The risk of end-stage renal disease (ESRD) remains high in patients with type 1diabetes and proteinuria; however, little is known about the rate of decline in their renal function. To help determine this we enrolled patients with 1 diabetes and proteinuria whose estimated glomerular filtration rate (eGFR) was normal (equal to or above 60 ml/min/1.73$m^2$). Using a minimum of 5 serial measurements of serum creatinine for 161 patients, we determined individual trajectories of eGFR change and the occurrence of ESRD during 5–18 years of follow-up. The rates were linear for 110 patients, for 24 the non-linear rate was mild enough to satisfy a linear model, and the rates were clearly non-linear for only 27 patients. Overall, in more than one third of patients, the eGFR decline was less than 3.5 ml/min/1.73$m^2$ per year and the lifetime risk of ESRD could be considered negligible. In the remainder of patients, eGFR declined with widely different slopes and ESRD developed within 2 to 18 years. Based on up to five years observation when renal function was within the normal range, the estimates of early eGFR slope predicted the risk of ESRD during subsequent follow-up better than the baseline clinical characteristics of glycated hemoglobin, blood pressure, or the albumin to creatinine ratio. Thus, the early slope of eGFR decline in patients with type 1diabetes and proteinuria can be used to predict the risk of ESRD