Evaluation of phylogenetic reconstruction methods using bacterial whole genomes: a simulation based study

Background: Phylogenetic reconstruction is a necessary first step in many analyses which use whole genome sequence data from bacterial populations. There are many available methods to infer phylogenies, and these have various advantages and disadvantages, but few unbiased comparisons of the range of approaches have been made. Methods: We simulated data from a defined "true tree" using a realistic evolutionary model. We built phylogenies from this data using a range of methods, and compared reconstructed trees to the true tree using two measures, noting the computational time needed for different phylogenetic reconstructions. We also used real data from Streptococcus pneumoniae alignments to compare individual core gene trees to a core genome tree. Results: We found that, as expected, maximum likelihood trees from good quality alignments were the most accurate, but also the most computationally intensive. Using less accurate phylogenetic reconstruction methods, we were able to obtain results of comparable accuracy; we found that approximate results can rapidly be obtained using genetic distance based methods. In real data we found that highly conserved core genes, such as those involved in translation, gave an inaccurate tree topology, whereas genes involved in recombination events gave inaccurate branch lengths. We also show a tree-of-trees, relating the results of different phylogenetic reconstructions to each other. Conclusions: We recommend three approaches, depending on requirements for accuracy and computational time. Quicker approaches that do not perform full maximum likelihood optimisation may be useful for many analyses requiring a phylogeny, as generating a high quality input alignment is likely to be the major limiting factor of accurate tree topology. We have publicly released our simulated data and code to enable further comparisons

Effectiveness of Residency Training Programs for Increasing Confidence and Competence among New Graduate Nurse Practitioners

Background: Nurse practitioners (NPs) are enrolling in post-graduate residency programs that provide training and mentorship during transition to practice. Objective: This project explored whether participation in NP residency improved feelings of confidence, competence, preparedness to provide care and job satisfaction among new graduate NPs who completed a residency program compared to new graduate NPs who did not complete a residency program. Method: This mixed-methods study collected survey data from NPs who attended the 2017 AANP National Conference and two residency programs. A modified Misener Nurse Practitioner Job Satisfaction Scale (MNPJSS) (Misener & Cox, 2001) and Hart’s New Nurse Practitioner Preparedness for Practice Survey (Hart & Bowen, 2016) were utilized. This survey contained 74-items rated on a Likert scale and three open-ended questions evaluating the stated objective. The results were entered into SPSS 25 software and analyzed using descriptive statistics. Results: There were significant differences in resident NPs’ preparedness to practice scores compared with NPs who did not complete residency. There was no difference in competence or job satisfaction scores between the two groups. This study also found a significant difference in those NPs who graduated from Doctoral NP (DNP) programs compared to Master in Science NP (MSN) programs in preparedness to practice, competence and one area of job satisfaction. Conclusions: This study supports DNP programs to facilitate transition to practice that improve outcomes in NP competence and overall preparedness to practice

16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice

Acknowledgements The authors acknowledge the assistance of Grietje Holtrop (RINH-BioSS) with the statistical analysis of the data and the Wellcome Trust Sanger Institute’s 454 pyrosequencing team for generating 16S rRNA gene data. AWW, PS and JP received core funding support from the Wellcome Trust [grant number 098051]. AWW, JCM, HJF and KPS are funded by the Scottish Government (SG-RESAS).Peer reviewedPublisher PD

Creating a national citizen engagement process for energy policy.

This paper examines some of the science communication challenges involved when designing and conducting public deliberation processes on issues of national importance. We take as our illustrative case study a recent research project investigating public values and attitudes toward future energy system change for the United Kingdom. National-level issues such as this are often particularly difficult to engage the public with because of their inherent complexity, derived from multiple interconnected elements and policy frames, extended scales of analysis, and different manifestations of uncertainty. With reference to the energy system project, we discuss ways of meeting a series of science communication challenges arising when engaging the public with national topics, including the need to articulate systems thinking and problem scale, to provide balanced information and policy framings in ways that open up spaces for reflection and deliberation, and the need for varied methods of facilitation and data synthesis that permit access to participants' broader values. Although resource intensive, national-level deliberation is possible and can produce useful insights both for participants and for science policy.Natural Environment Research CouncilLeverhulme TrustWelsh GovernmentUS National Science Foundatio

A non-endoscopic device to sample the oesophageal microbiota: a case-control study.

BACKGROUND: The strongest risk factor for oesophageal adenocarcinoma is reflux disease, and the rising incidence of this coincides with the eradication of Helicobacter pylori, both of which might alter the oesophageal microbiota. We aimed to profile the microbiota at different stages of Barrett's carcinogenesis and investigate the Cytosponge as a minimally invasive tool for sampling the oesophageal microbiota. METHODS: In this case-control study, 16S rRNA gene amplicon sequencing was done on 210 oesophageal samples from 86 patients representing the Barrett's oesophagus progression sequence (normal squamous controls [n=20], non-dysplastic [n=24] and dysplastic Barrett's oesophagus [n=23], and oesophageal adenocarcinoma [n=19]), relevant negative controls, and replicates on the Illumina MiSeq platform. Samples were taken from patients enrolled in the BEST2 study at five UK hospitals and the OCCAMS study at six UK hospitals. We compared fresh frozen tissue, fresh frozen endoscopic brushings, and the Cytosponge device for microbial DNA yield (qPCR), diversity, and community composition. FINDINGS: There was decreased microbial diversity in oesophageal adenocarcinoma tissue compared with tissue from healthy control patients as measured by the observed operational taxonomic unit (OTU) richness (p=0·0012), Chao estimated total richness (p=0·0004), and Shannon diversity index (p=0·0075). Lactobacillus fermentum was enriched in oesophageal adenocarcinoma (p=0·028), and lactic acid bacteria dominated the microenvironment in seven (47%) of 15 cases of oesophageal adenocarcinoma. Comparison of oesophageal sampling methods showed that the Cytosponge yielded more than ten-times higher quantities of microbial DNA than did endoscopic brushes or biopsies using quantitative PCR (p<0·0001). The Cytosponge samples contained the majority of taxa detected in biopsy and brush samples, but were enriched for genera from the oral cavity and stomach, including Fusobacterium, Megasphaera, Campylobacter, Capnocytophaga, and Dialister. The Cytosponge detected decreased microbial diversity in patients with high-grade dysplasia in comparison to control patients, as measured by the observed OTU richness (p=0·0147), Chao estimated total richness (p=0·023), and Shannon diversity index (p=0·0085). INTERPRETATION: Alterations in microbial communities occur in the lower oesophagus in Barrett's carcinogenesis, which can be detected at the pre-invasive stage of high-grade dysplasia with the novel Cytosponge device. Our findings are potentially applicable to early disease detection, and future test development should focus on longitudinal sampling of the microbiota to monitor for changes in microbial diversity in a larger cohort of patients. FUNDING: Cancer Research UK, National Institute for Health Research, Medical Research Council, Wellcome Trust, The Scottish Government (RESAS).Cancer Research UK, National Institute for Health Research, Medical Research Council, and Wellcome Trust.This is the author accepted manuscript. The final version is available from Elsevier via https://doi.org/10.1016/S2468-1253(16)30086-

Evaluation of PacBio sequencing for full-length bacterial 16S rRNA gene classification.

BACKGROUND: Currently, bacterial 16S rRNA gene analyses are based on sequencing of individual variable regions of the 16S rRNA gene (Kozich, et al Appl Environ Microbiol 79:5112-5120, 2013).This short read approach can introduce biases. Thus, full-length bacterial 16S rRNA gene sequencing is needed to reduced biases. A new alternative for full-length bacterial 16S rRNA gene sequencing is offered by PacBio single molecule, real-time (SMRT) technology. The aim of our study was to validate PacBio P6 sequencing chemistry using three approaches: 1) sequencing the full-length bacterial 16S rRNA gene from a single bacterial species Staphylococcus aureus to analyze error modes and to optimize the bioinformatics pipeline; 2) sequencing the full-length bacterial 16S rRNA gene from a pool of 50 different bacterial colonies from human stool samples to compare with full-length bacterial 16S rRNA capillary sequence; and 3) sequencing the full-length bacterial 16S rRNA genes from 11 vaginal microbiome samples and compare with in silico selected bacterial 16S rRNA V1V2 gene region and with bacterial 16S rRNA V1V2 gene regions sequenced using the Illumina MiSeq. RESULTS: Our optimized bioinformatics pipeline for PacBio sequence analysis was able to achieve an error rate of 0.007% on the Staphylococcus aureus full-length 16S rRNA gene. Capillary sequencing of the full-length bacterial 16S rRNA gene from the pool of 50 colonies from stool identified 40 bacterial species of which up to 80% could be identified by PacBio full-length bacterial 16S rRNA gene sequencing. Analysis of the human vaginal microbiome using the bacterial 16S rRNA V1V2 gene region on MiSeq generated 129 operational taxonomic units (OTUs) from which 70 species could be identified. For the PacBio, 36,000 sequences from over 58,000 raw reads could be assigned to a barcode, and the in silico selected bacterial 16S rRNA V1V2 gene region generated 154 OTUs grouped into 63 species, of which 62% were shared with the MiSeq dataset. The PacBio full-length bacterial 16S rRNA gene datasets generated 261 OTUs, which were grouped into 52 species, of which 54% were shared with the MiSeq dataset. Alpha diversity index reported a higher diversity in the MiSeq dataset. CONCLUSION: The PacBio sequencing error rate is now in the same range of the previously widely used Roche 454 sequencing platform and current MiSeq platform. Species-level microbiome analysis revealed some inconsistencies between the full-length bacterial 16S rRNA gene capillary sequencing and PacBio sequencing

Meningococcal genetic variation mechanisms viewed through comparative analysis of Serogroup C strain FAM18

Copyright @ 2007 Public Library of ScienceThe bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.This work was supported by the Wellcome Trust through the Beowulf Genomics Initiative