Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

determination-microbial-biomass-soil-using-digital-droplet-pcr

Determination of Microbial Biomass in Soil using Digital Droplet PCR (ddPCR)

Soil represents the largest terrestrial pool of organic carbon, yet more than 133 Gt of carbon has been lost from soils over the past two centuries. ...

University of Exeter

Vaccines 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.

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

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.

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

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.

Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii.

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 virus 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 virus 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 a certain level of 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.

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

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.

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

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that  11168H forms persister cells at a frequency of 10 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated  cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

 has potential as an industrial biocatalyst due to its ability to produce lactic acid. A novel strain of  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.

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

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.

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

Atmospheric particulate matter (PM) causes 3.7 million annual deaths worldwide and potentially damages every organ in the body. The cancer-causing potential of fine particulates (PM) highlights the inextricable link between air quality and human health. With over half of the world's population living in cities, PM emissions are a major concern, however, our understanding of exposure to urban PM is restricted to relatively recent (post-1990) air quality monitoring programmes. To investigate how the composition and toxicity of PM has varied within an urban region, over timescales encompassing changing patterns of industrialisation and urbanisation, we reconstructed air pollution records spanning 200 years from the sediments of urban ponds in Merseyside (NW England), a heartland of urbanisation since the Industrial Revolution. These archives of urban environmental change across the region demonstrate a key shift in PM emissions from coarse carbonaceous 'soot' that peaked during the mid-twentieth century, to finer combustion-derived PM post-1980, mirroring changes in urban infrastructure. The evolution of urban pollution to a recent enhanced PM signal has important implications for understanding lifetime pollution exposures for urban populations over generational timescales.

The evolution of atmospheric particulate matter in an urban landscape since the Industrial Revolution.

Anaerobic digestion is an established method for the biological conversion of waste feedstocks to biogas and biomethane. While anaerobic digestion is an excellent waste management technique, it can be susceptible to toxins and pollutants from contaminated feedstocks, which may have a detrimental impact on a digester's efficiency and productivity. Ethylene glycol (EG) is readily used in the heat-transfer loops of anaerobic digestion facilities to maintain reactor temperature. Failure of the structural integrity of these heat transfer loops can cause EG to leak into the digester, potentially causing a decrease in the resultant gas yields. Batch fermentations were incubated with 0, 10, 100 and 500 ppm (parts per million) of EG, and analysis showed that the EG was completely metabolised by the digester microbiome. The concentrations of EG tested showed significant increases in gas yields, however there were no significant changes to the digester microbiome.

Metagenomic analysis of ethylene glycol contamination in anaerobic digestion.

Pollen and tracheophyte spores are ubiquitous environmental indicators at local and global scales. Palynology is typically performed manually by microscopic analysis; a specialised and time-consuming task limited in taxonomical precision and sampling frequency, therefore restricting data quality used to inform climate change and pollen forecasting models. We build on the growing work using AI (artificial intelligence) for automated pollen classification to design a flexible network that can deal with the uncertainty of broad-scale environmental applications. We combined imaging flow cytometry with Guided Deep Learning to identify and accurately categorise pollen in environmental samples; here, pollen grains captured within c. 5500 Cal yr BP old lake sediments. Our network discriminates not only pollen included in training libraries to the species level but, depending on the sample, can classify previously unseen pollen to the likely phylogenetic order, family and even genus. Our approach offers valuable insights into the development of a widely transferable, rapid and accurate exploratory tool for pollen classification in 'real-world' environmental samples with improved accuracy over pure deep learning techniques. This work has the potential to revolutionise many aspects of palynology, allowing a more detailed spatial and temporal understanding of pollen in the environment with improved taxonomical resolution.

Deductive automated pollen classification in environmental samples via exploratory deep learning and imaging flow cytometry.

Metagenomics has been transformative in our understanding of the diversity and function of soil microbial communities. Applying long read sequencing to whole genome shotgun metagenomics has the potential to revolutionise soil microbial ecology through improved taxonomic classification, functional characterisation and metagenome assembly. However, optimisation of robust methods for long read metagenomics of environmental samples remains undeveloped. In this study, Oxford Nanopore sequencing using samples from five commercially available soil DNA extraction kits was compared across four soil types, in order to optimise read length and reproducibility for comparative long read soil metagenomics. Average extracted DNA lengths varied considerably between kits, but longer DNA fragments did not translate consistently into read lengths. Highly variable decreases in the length of resulting reads from some kits were associated with poor classification rate and low reproducibility in microbial communities identified between technical repeats. Replicate samples from other kits showed more consistent conversion of extracted DNA fragment size into read length and resulted in more congruous microbial community representation. Furthermore, extraction kits showed significant differences in the community representation and structure they identified across all soil types. Overall, the QIAGEN DNeasy PowerSoil Pro Kit displayed the best suitability for reproducible long-read WGS metagenomic sequencing, although further optimisation of DNA purification and library preparation may enable translation of higher molecular weight DNA from other kits into longer read lengths. These findings provide a novel insight into the importance of optimising DNA extraction for achieving replicable results from long read metagenomic sequencing of environmental samples.

Comparative evaluation of soil DNA extraction kits for long read metagenomic sequencing.

Metagenomic analysis has enabled insights into soil community structure and dynamics. Long-read sequencing for metagenomics can enhance microbial ecology by improving taxonomic classification, genome assembly, and functional annotation. However, protocols for purifying high-molecular weight DNA from soil are not yet optimised. We used a statistical design of experiments approach to enhance mechanical lysis of soil samples, increasing the length of purified DNA fragments. Low energy input into mechanical lysis improved DNA integrity, resulting in longer sequenced reads. Our optimized settings of 4 m s for 10 s increased fragment length by 70% compared to the manufacturer's recommendations. Longer reads from low intensity lysis produced longer contiguous sequences after assembly, potentially improving a range of down-stream analyses. Importantly, there was minimal bias exhibited in the microbial community composition due to lysis efficiency variations. We therefore propose a framework for improving the fragment lengths of DNA purified from diverse soil types, improving soil science research with long-read sequencing.

Statistical design approach enables optimised mechanical lysis for enhanced long-read soil metagenomics.

Direct sequencing of DNA from environmental samples (eDNA) is increasingly utilized to provide a census of natural and industrial habitats. The methodology required to perform metagenomics can be divided into three distinct stages: DNA Purification, Library Preparation and Sequencing, and Bioinformatic Analysis. Here we demonstrate an end-to-end protocol that can be utilized either in the field or laboratory for metagenomic analysis of environmental samples utilizing the Oxford Nanopore Technologies MinION sequencing platform.

Laboratory and In-Field Metagenomics for Environmental Monitoring.

Long-read sequencing has revolutionised metagenomics through improved metagenome assembly, taxonomic classification and functional characterisation. Automation can enhance the throughput, reproducibility, and accuracy of library preparation. However, the validation of automated library preparation protocols remains undetermined for metagenomic workflows, which are particularly sensitive to methodological perturbation. Here, we compare long-read metagenomic sequencing of environmental samples through parallel manual and automated protocols.

ArticleTotal : 2	Year
Publication title Cited by 19	2017
Publication title	2026

Article	Year
Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory. eLife\| PubMed ID: 29709214	2018
A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200. Microbiology resource announcements\| PubMed ID: 30687838	2019
Palaeogenomics of the Hydrocarbon Producing Microalga Botryococcus braunii. Scientific reports\| PubMed ID: 30742038	2019
Higher levels of B-cell mutation in the early germinal centres of an inefficient secondary antibody response to a variant influenza haemagglutinin. Immunology\| PubMed ID: 30768794	2019
Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications. ACS synthetic biology\| PubMed ID: 30995831	2019
11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity. Frontiers in cellular and infection microbiology\| PubMed ID: 33194805	2020
Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival. PLoS pathogens\| PubMed ID: 33439894	2021
Draft Genome Sequence of Weissella paramesenteroides STCH-BD1, Isolated from Ensiled Sorghum bicolor. Microbiology resource announcements\| PubMed ID: 33602733	2021
In-situ sequencing reveals the effect of storage on lacustrine sediment microbiome demographics and functionality. Environmental microbiome\| PubMed ID: 35101122	2022
The evolution of atmospheric particulate matter in an urban landscape since the Industrial Revolution. Scientific reports\| PubMed ID: 37268751	2023
Metagenomic analysis of ethylene glycol contamination in anaerobic digestion. Bioresource technology\| PubMed ID: 37597572	2023
Deductive automated pollen classification in environmental samples via exploratory deep learning and imaging flow cytometry. The New phytologist\| PubMed ID: 37678361	2023
Comparative evaluation of soil DNA extraction kits for long read metagenomic sequencing. Access microbiology\| PubMed ID: 39346682	2024
Statistical design approach enables optimised mechanical lysis for enhanced long-read soil metagenomics. Scientific reports\| PubMed ID: 39578630	2024
Laboratory and In-Field Metagenomics for Environmental Monitoring. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 40736894	2025
Automated environmental metagenomics using Oxford nanopore sequencing. BMC genomics\| PubMed ID: 41013192	2025

Richard K. Tennant

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