CowPI::A rumen microbiome focussed version of the PICRUSt functional inference software

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and over 500 fully sequenced microbial genomes from this environment. Using this information we have developed ?CowPI? a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and captures the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiomepublishersversionPeer reviewe

The importance of extracellular vesicle purification for downstream analysis:A comparison of differential centrifugation and size exclusion chromatography for helminth pathogens

BackgroundRobust protocols for the isolation of extracellular vesicles (EVs) from the rest of their excretory-secretory products are necessary for downstream studies and application development. The most widely used purification method of EVs for helminth pathogens is currently differential centrifugation (DC). In contrast, size exclusion chromatography (SEC) has been included in the purification pipeline for EVs from other pathogens, highlighting there is not an agreed research community ‘gold standard’ for EV isolation. In this case study, Fasciola hepatica from natural populations were cultured in order to collect EVs from culture media and evaluate a SEC or DC approach to pathogen helminth EV purification.Methodology/Principal findingsTransmission electron and atomic force microscopy demonstrated that EVs prepared by SEC were both smaller in size and less diverse than EV resolved by DC. Protein quantification and Western blotting further demonstrated that SEC purification realised a higher EV purity to free excretory-secretory protein (ESP) yield ratio compared to DC approaches as evident by the reduction of soluble free cathepsin L proteases in SEC EV preparations. Proteomic analysis further highlighted DC contamination from ESP as shown by an increased diversity of protein identifications and unique peptide hits in DC EVs as compared to SEC EVs. In addition, SEC purified EVs contained less tegumental based proteins than DC purified EVs.Conclusions/SignificanceThe data suggests that DC and SEC purification methods do not isolate equivalent EV population profiles and caution should be taken in the choice of EV purification utilised, with certain protocols for DC preparations including more free ES proteins and tegumental artefacts. We propose that SEC methods should be used for EV purification prior to downstream studies.</div

Relational care and co-operative endeavour:Reshaping dementia care through participatory secondary data analysis

Dementia is emerging from the shadows of societal exclusion and stigma. The engagement within society for people who are marginalised is coconstructed through the everyday practices that take place between them and those around them. However, this is inherently political, positioning people as active and activist in the relationship of their lives with their communities. The research aimed to interrogate an existing qualitative dataset in partnership with people living with dementia to inform the development of a way of working with people with dementia that is empowering. In this qualitative secondary data analysis project, we (1) analysed data through two theoretical lenses: Douglas’ cultural theory of risk and Tronto’s Ethic of Care, and (2) co-analysed the data together with people living with dementia during 14 workshops. The design involved cycles of presenting, interpreting, representing and reinterpreting the data and findings between multiple stakeholders. We identified a granular understanding of the way relationships change for people with dementia and how subtle factors and nuanced behaviour contribute to social exclusion, or support social inclusion. The results support relational care through the co-operative endeavour (of co-operative communication, cooperative action and co-operative care) in promoting the inclusion of people living with dementia

Using next-generation sequencing to determine diversity of horse intestinal worms:Identifying the equine ʼnemabiome'

Next generation sequencing of DNA from nematode eggs has been utilised to give the first account of the equine ‘nemabiome’. In all equine faecal samples investigated, multiple species of Strongylidae were detected; ranging from 7.5 (SEM 0.79) with 99+% identity to sequences in the NCBI database to 13.3 (SEM 0.80) with 90+% identity. This range is typical of the number of species described previously in morphological studies using large quantities of digesta per animal. However, the current method is non-invasive, relies on DNA analysis avoiding the need for specialist microscopy identification and can be carried out with small samples providing significant advantages over current methods