Metagenomic analysis of silage

This is the final version of the article. Available from the publisher via the DOI in this record.Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.Authors would like to thank Andrew Bird for the silage samples and Audrey Farbos of the Exeter Sequencing Service for her assistance in preparing DNA sequencing libraries. Exeter Sequencing Service and Computational core facilities at the University of Exeter. Medical Research Council Clinical Infrastructure award (MR/M008924/1). Wellcome Trust Institutional Strategic Support Fund (WT097835MF), Wellcome Trust Multi User Equipment Award (WT101650MA) and BBSRC LOLA award (BB/K003240/1

Higher levels of B-cell mutation in the early germinal centres of an inefficient secondary antibody response to a variant Influenza Haemagglutinin

This is the final version. Available from the publisher via the DOI in this record.Designing improved vaccines against mutable viruses such as Dengue and Influenza would be helped by a better understanding of how the B-cell memory compartment responds to variant antigens. Towards this we have recently shown after secondary immunization of mice with a widely variant Dengue envelope protein, with only 63% amino-acid identity, that IgM+ memory B-cells with few mutations supported an efficient secondary germinal centre (GC) and serum response, superior to a primary response to the same protein. Here, further investigation of memory responses to variant proteins, using more closely related Influenza haemagglutinins (HA), that were 82% identical, produced a variant-induced boost response in the GC dominated by highly mutated B-cells that failed, not efficiently improving serum avidity even in the presence of extra adjuvant, and that was worse than a primary response. This supports a hypothesis that over certain antigenic differences, cross-reactive memory B-cell populations have reduced competency for affinity maturation. Combined with our previous observations these findings also provide new parameters of success and failure in antibody memory responses. This article is protected by copyright. All rights reserved.Wellcome TrustBiotechnology and Biological Sciences Research Council (BBSRC

Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory

This is the final version. Available from eLife Sciences Publications via the DOI in this recordVaccines induce memory B-cells that provide high affinity secondary antibody responses to identical antigens. Memory B-cells can also re-instigate affinity maturation, but how this happens against antigenic variants is poorly understood despite its potential impact on driving broadly protective immunity against pathogens such as Influenza and Dengue. We immunised mice sequentially with identical or variant Dengue-virus envelope proteins and analysed antibody and germinal-centre (GC) responses. Variant protein boosts induced GCs with a higher proportion of IgM+ B cells. The most variant protein re-stimulated GCs with the highest proportion of IgM+ cells with the most diverse, least mutated V-genes and with a slower but efficient serum antibody response. Recombinant antibodies from GC B-cells showed a higher affinity for the variant antigen than antibodies from a primary response, confirming a memory origin. This reveals a new process of antibody memory, that IgM memory cells with fewer mutations participate in secondary responses to variant antigens, demonstrating how the hierarchical structure of B-cell memory is used and indicating the potential and limits of cross-reactive antibody based immunity.Funder: Wellcome Trust; Grant reference number 100115/Z/12/

Monitoring Impacts of Urbanisation and Industrialisation on Air Quality in the Anthropocene Using Urban Pond Sediments

This is the final version of the article. Available from Frontiers Media via the DOI in this record.The release of toxic atmospheric pollutants since the Industrial Revolution is a major global challenge, driving climate change and damaging human health. Spatial health inequalities highlight the need to explore air pollution in different localities throughout the Anthropocene. Air quality monitoring programmes are spatially and temporally limited. We show how suitable urban sediment archives provide site specific records of long-term particulate matter (PM) releases, in cities and urban landscapes, that are or have been subjected to high pollution levels. High-resolution PM records spanning from the mid-20th century were reconstructed from an urban pond in Chongqing, southwest China, one of the fastest growing Chinese urban centres in the late 20th century. Temporal variations in pollution proxies including geomagnetic, geochemical and spheroidal carbonaceous particle trends correspond to key phases of industrial and urban developments, that are representative of the locality. Observed increases in air pollution proxies post-1960 coincide with the location of military-related industries to Chongqing and industrial intensification. Post-1997 pollution mirrors rapid urbanisation that occurred following the designation of Chongqing as a government-controlled municipality at this time and reveals a steadily increasing pollution trend to present day (2015). In comparison to Chongqing, an atmospheric depositional history was constructed from an urban pond in the Merseyside region of northwest England that has experienced a legacy of contamination since the early 19th century. In northwest England, changing characteristics of pollution are related to the establishment of localised, modern industries, power generation, urban sprawl, increased combustion-derived pollution post-1990 and effective pollution legislations. Whereas a reduction in air pollution has occurred post-2000 in Merseyside, in Chongqing, however, air pollution has continued to increase in spite of national efforts in pollution control. These urban sediments reveal the changing nature of air pollution in different urban landscapes, allowing us to assess air quality impacts of progressive industrial activity, increased road and air travel, urban expansion and the efficacy of pollution controls. It appears that air pollution remains an inevitable consequence of global industrialisation. It is therefore crucial to understand pollution histories in densely populated urban regions to determine environmental burdens of pollution on health over generational timescales.This research was supported by Halton Primary Care Trust (grant number CHCRD001 SE02), Edge Hill University (fund codes: RDWOR04, RDWOR05, RDWOR06, and RDWOR209), and the University of Exeter

Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications

This is the author accepted manusript. The final version is available from American Chemical Society via the DOI in this recordAccession Codes: The sequence data for the four Geobacillus spp. used in this study have been submitted to the NCBI Sequence Read Archive and are available under the accession number PRJNA521450.Well-characterized promoter collections for synthetic biology applications are not always available in industrially relevant hosts. We developed a broadly applicable method for promoter identification in atypical microbial hosts that requires no a priori understanding of cis-regulatory element structure. This novel approach combines bioinformatic filtering with rapid empirical characterization to expand the promoter toolkit and uses machine learning to improve the understanding of the relationship between DNA sequence and function. Here, we apply the method in Geobacillus thermoglucosidasius, a thermophilic organism with high potential as a synthetic biology chassis for industrial applications. Bioinformatic screening of G. kaustophilus, G. stearothermophilus, G. thermodenitrificans, and G. thermoglucosidasius resulted in the identification of 636 100 bp putative promoters, encompassing the genome-wide design space and lacking known transcription factor binding sites. Eighty of these sequences were characterized in vivo, and activities covered a 2-log range of predictable expression levels. Seven sequences were shown to function consistently regardless of the downstream coding sequence. Partition modeling identified sequence positions upstream of the canonical -35 and -10 consensus motifs that were predicted to strongly influence regulatory activity in Geobacillus, and artificial neural network and partial least squares regression models were derived to assess if there were a simple, forward, quantitative method for in silico prediction of promoter function. However, the models were insufficiently general to predict pre hoc promoter activity in vivo, most probably as a result of the relatively small size of the training data set compared to the size of the modeled design space

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200

This is the final version. Available on open access from American Society for Microbiology via the DOI in this recordData availability.The complete genome sequence of T. ethanolicus JW 200 is deposited in GenBank under the accession number CP033580. Illumina and Oxford Nanopore DNA sequence reads have been deposited in the NCBI Sequence Read Archive (accession numbers SRR8113455 and SRR8113456).Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp.Shell Research Ltd

Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii

This is the final version. Available from the publisher via the DOI in this record.Botryococcus braunii is a colonial microalga that appears early in the fossil record and is a sensitive proxy of environmental and hydroclimatic conditions. Palaeozoic Botryococcus fossils which contribute up to 90% of oil shales and approximately 1% of crude oil, co-localise with diagnostic geolipids from the degradation of source-signature hydrocarbons. However more recent Holocene sediments demonstrate no such association. Consequently, Botryococcus are identifed in younger sediments by morphology alone, where potential misclassifcations could lead to inaccurate paleoenvironmental reconstructions. Here we show that a combination of fow cytometry and ancient DNA (aDNA) sequencing can unambiguously identify Botryococcus microfossils in Holocene sediments with hitherto unparalleled accuracy and rapidity. The application of aDNA sequencing to microfossils ofers a far-reaching opportunity for understanding environmental change in the recent geological record. When allied with other high-resolution palaeoenvironmental information such as aDNA sequencing of humans and megafauna, aDNA from microfossils may allow a deeper and more precise understanding of past environments, ecologies and migrations.Medical Research Council (MRC)Wellcome TrustBiotechnology and Biological Science Research Council (BBSRC

Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii.

Botryococcus braunii is a colonial microalga that appears early in the fossil record and is a sensitive proxy of environmental and hydroclimatic conditions. Palaeozoic Botryococcus fossils which contribute up to 90% of oil shales and approximately 1% of crude oil, co-localise with diagnostic geolipids from the degradation of source-signature hydrocarbons. However more recent Holocene sediments demonstrate no such association. Consequently, Botryococcus are identified in younger sediments by morphology alone, where potential misclassifications could lead to inaccurate paleoenvironmental reconstructions. Here we show that a combination of flow cytometry and ancient DNA (aDNA) sequencing can unambiguously identify Botryococcus microfossils in Holocene sediments with hitherto unparalleled accuracy and rapidity. The application of aDNA sequencing to microfossils offers a far-reaching opportunity for understanding environmental change in the recent geological record. When allied with other high-resolution palaeoenvironmental information such as aDNA sequencing of humans and megafauna, aDNA from microfossils may allow a deeper and more precise understanding of past environments, ecologies and migrations

In-situ sequencing reveals the effect of storage on lacustrine sediment microbiome demographics and functionality

This is the final version. Available from BMC via the DOI in this record. Availability of data and materials: The datasets generated and analysed during the current study are available in the NCBI SRA repository under BioProject ID PRJNA548524.The sediment microbiome is a demographically diverse and functionally active biosphere. Ensuring that data acquired from sediment is truly representative of the microbiome is critical to achieving robust analyses. Sample storage and the processing and timing of nucleic acid purification after environmental sample extraction may fundamentally affect the detectable microbial community and thereby significantly alter resultant data. Direct sequencing of environmental samples is increasingly commonplace due to the advent of the portable Oxford Nanopore MinION sequencing device. Here we demonstrate that storing sediment subsamples at −20 °C or storing the cores at 4 °C for 10 weeks prior to analysis, has a significant effect on the sediment microbiome analysed using sedimentary DNA (sedDNA), especially for Alpha-, Beta- and Deltaproteobacteria species. Furthermore, these significant differences are observed regardless of sediment type. We show that the taxa which are predominantly affected by storage are Proteobacteria, and therefore recommend on-site purifications are performed to ensure an accurate representation of these taxa are observed in the microbiome. Comparisons of sedimentary RNA (sedRNA) analyses, revealed substantial differences between samples purified and sequenced immediately on-site, samples that were frozen before transportation, and cores that were stored at 4 °C prior to analysis. Our data therefore suggest that a more accurate representation of the sediment microbiome demography and functionality may be achieved by environmental sequencing as rapidly as possible to minimise confounding effects of storage.University of Exete

Draft Genome Sequence of Weissella paramesenteroides STCH-BD1, Isolated from Ensiled Sorghum bicolor

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordData availability. The draft genome sequence of W. paramesenteroides STCH-BD1 is deposited in GenBank under the accession numbers CP065045 and CP065046. Oxford Nanopore and Illumina DNA sequence reads have been deposited in the NCBI Sequence Read Archive under accession numbers SRR13083241 and SRR13083242, respectivelyWeissella paramesenteroides has potential as an industrial biocatalyst due to its ability to produce lactic acid. A novel strain of W. paramesenteroides was isolated from ensiled sorghum. The genome was sequenced using a hybrid assembly of Oxford Nanopore and Illumina data to produce a 2-Mbp genome and 22-kbp plasmid sequence.Shell International Exploration and Production, In