In this thesis I highlight the applications of Oxford Nanopore Technologies (ONT) sequencing. This technique is a relatively new approach in the sequencing field, where nanopores are embedded in a... Show moreIn this thesis I highlight the applications of Oxford Nanopore Technologies (ONT) sequencing. This technique is a relatively new approach in the sequencing field, where nanopores are embedded in a membrane, DNA molecules are pulled through nanopores and an electrical current serving as the sequencing signal. This technique yields reads-lengths of >10Kbp and has no theoretical upper limit towards read-length. The positive impact on data quality due to improved chemistry is underlined, improved chemistry leads to less sequencing errors and a more homogeneous coverage over complex genomic architectures. Benefits for increased read-lengths are assessed for resolving fragmented genome assemblies that were previously based solely on short-read sequencing data. Furthermore, the assembly of a large genome using ONT data is described, indicating ONT is a suitable candidate for resolving extremely large genomes using sophisticated assembly approaches. And finally, the potential for on-site sequencing is evaluated. Exploiting simplicity, mobility and accuracy provided by this new technique. The central hypothesis of this thesis is that Oxford Nanopore Technologies long-read sequencing can be valuable for established genomics applications, such as whole genome sequencing and metagenomic characterization of microbial communities. Show less
Metagenomics enables the detection of all the genetic material of organisms present in a sample, making it a pathogen-agnostic approach for detecting common and rare or novel pathogens that are not... Show moreMetagenomics enables the detection of all the genetic material of organisms present in a sample, making it a pathogen-agnostic approach for detecting common and rare or novel pathogens that are not included in conventional testing. Beforehand, a clinician does not need to have a hypothesis of what pathogen is expected, unlike traditional polymerase chain reaction (PCR) testing.This thesis is focusing on diagnostic yield, clinical findings, and enhancing technical opportunities in viral metagenomics. The identification, typing, and quantification of viruses by means of viral metagenomics as a diagnostic tool are evaluated. Technical aspects are appraised for improved sensitivity and specificity of the wet and dry (bioinformatic) lab components of viral metagenomics. The use of a metagenomic protocol for virus discovery directly in a patient sample is assessed, and the best methods and approaches for performing genetic analysis of the SARS-CoV-2 virus are investigated.Viral metagenomic testing results in the identification of more viruses, therefore it is a valuable addition to current diagnostic test protocols. Additionally, it is a useful test for virus discovery and monitoring during infectious disease outbreaks caused by novel viruses. Show less
Borry, M.; Forsythe, A.; Valtueña, A.A.; Hübner, A.; Ibrahim, A.; Quagliariello, A; ... ; Fellows Yates, J.A. 2023
Background: Access to sample-level metadata is important when selecting public metagenomic sequencing datasets for reuse in new biological analyses. The Standards, Precautions, and Advances in... Show moreBackground: Access to sample-level metadata is important when selecting public metagenomic sequencing datasets for reuse in new biological analyses. The Standards, Precautions, and Advances in Ancient Metagenomics community (SPAAM, https://spaam-community.github.io) has previously published AncientMetagenomeDir, a collection of curated and standardised sample metadata tables for metagenomic and microbial genome datasets generated from ancient samples. However, while sample-level information is useful for identifying relevant samples for inclusion in new projects, Next Generation Sequencing (NGS) library construction and sequencing metadata are also essential for appropriately reprocessing ancient metagenomic data. Currently, recovering information for downloading and preparing such data is difficult when laboratory and bioinformatic metadata is heterogeneously recorded in prose-based publications. Methods: Through a series of community-based hackathon events, AncientMetagenomeDir was updated to provide standardised library-level metadata of existing and new ancient metagenomic samples. In tandem, the companion tool 'AMDirT' was developed to facilitate automated metadata curation and data validation, as well as rapid data filtering and downloading. Results: AncientMetagenomeDir was extended to include standardised metadata of over 5000 ancient metagenomic libraries. The companion tool 'AMDirT' provides both graphical- and command-line interface based access to such metadata for users from a wide range of computational backgrounds. We also report on errors with metadata reporting that appear to commonly occur during data upload and provide suggestions on how to improve the quality of data sharing by the community.Conclusions: Together, both standardised metadata and tooling will help towards easier incorporation and reuse of public ancient metagenomic datasets into future analyses. Show less
Fagernäs Z.; Salazar-García, D.C.; Haber Uriarte, M.; Avilés Fernández, A.; Henry A.G.; Lomba Maurandi, J.; ... ; Warinner, C. 2022
The oral cavity is a heterogeneous environment, varying in factors such as pH, oxygen levels, and salivary flow. These factors affect the microbial community composition and distribution of species... Show moreThe oral cavity is a heterogeneous environment, varying in factors such as pH, oxygen levels, and salivary flow. These factors affect the microbial community composition and distribution of species in dental plaque, but it is not known how well these patterns are reflected in archaeological dental calculus. In most archaeological studies, a single sample of dental calculus is studied per individual and is assumed to represent the entire oral cavity. However, it is not known if this sampling strategy introduces biases into studies of the ancient oral microbiome. Here, we present the results of a shotgun metagenomic study of a dense sampling of dental calculus from four Chalcolithic individuals from the southeast Iberian peninsula (ca. 4500–5000 BP). Interindividual differences in microbial composition are found to be much larger than intraindividual differences, indicating that a single sample can indeed represent an individual in most cases. However, there are minor spatial patterns in species distribution within the oral cavity that should be taken into account when designing a study or interpreting results. Finally, we show that plant DNA identified in the samples is likely of postmortem origin, demonstrating the importance of including environmental controls or additional lines of biomolecular evidence in dietary interpretations. Show less
Soil is a home for an unbelievable diversity and abundance of microbial life that is essential for supporting life on our planet. Microorganisms living in soil take part in cleaning our water,... Show moreSoil is a home for an unbelievable diversity and abundance of microbial life that is essential for supporting life on our planet. Microorganisms living in soil take part in cleaning our water, degrading toxic compounds and recycling nutrients, and last but not least, they are essential partners to plants. Through their roots, plants release a mix of secretions to attract microorganisms, creating a remarkable environment called the rhizosphere. The rhizosphere is populated by microbes who often provide beneficial services to plants, like nutrient acquisition, growth promotion and protection against diseases. Modern agriculture suffers from losses caused by crop diseases, and a common way of controlling diseases is using pesticides. Pesticides often have a negative impact on the environment, and disease-causing agents (pathogens), can become resistant with time. One of the possible solutions to this problem is based on soil microbial communities. Due to the activity of their microbiome, some soils possess a natural capacity to protect plants against diseases. These soils are called disease suppressive soils, and the investigation of the microbial mechanisms leading to the natural protection of crops is the topic of this thesis. In our work we used a common pathogen of cereals, fungus Fusarium culmorum, and wheat, to first, identify suppressive soils able to protect this plant from the pathogen, and, investigate the mechanisms of protection using e.g. sequencing and mass spectrometry. During our project we identified potential microbes, genes and metabolites involved in soil disease suppressiveness. Moreover, we evaluated the impact of microplastic on the soil disease suppressiveness. Show less
Acidobacteria is a widely distributed phylum but their functional roles in ecosystem processes are still largely elusive. The Granulicella genus belongs to the class Acidobacteriia and is known to... Show moreAcidobacteria is a widely distributed phylum but their functional roles in ecosystem processes are still largely elusive. The Granulicella genus belongs to the class Acidobacteriia and is known to produce copious amount of Extracellular Polymeric Substances (EPS), which are fundamental for microbial life. The major goal of my thesis was to investigate and understand the metabolism of Granulicella sp. strains WH15 and 5B5, and the functions and environmental fate of EPS of Granulicella sp. WH15 (WH15EPS). Optimization of carbon concentration and manganese in culture medium allowed our strains, especially WH15, to grow faster in laboratory conditions, producing extractable amounts of EPS. Using the Stable Isotope Probing technique, we observed the incorporation of WH15EPS by Singulisphaera and its connections to other Planctomycetes and Acidobacteria, which were not reported before. In addition, our results showed, in the genomes of the microbes which incorporated WH15EPS, a large diversity of glycoside hydrolase with biotechnological potential and a high number of unclassified microorganisms that could be targeted for future studies. The data presented in this thesis establish a solid fundamental basis for more mechanistic studies of Acidobacteria and other uncultivated microbes. Show less
The assessment of microbiome biodiversity is the most common application of metagenomics. While 16S sequencing remains standard procedure for taxonomic profiling of metagenomic data, a growing... Show moreThe assessment of microbiome biodiversity is the most common application of metagenomics. While 16S sequencing remains standard procedure for taxonomic profiling of metagenomic data, a growing number of studies have clearly demonstrated biases associated with this method. By using Whole Genome Shotgun sequencing (WGS) metagenomics, most of the known restrictions associated with 16S data are alleviated. However, due to the computationally intensive data analyses and higher sequencing costs, WGS based metagenomics remains a less popular option. Selecting the experiment type that provides a comprehensive, yet manageable amount of information is a challenge encountered in many metagenomics studies. In this work, we created a series of artificial bacterial mixes, each with a different distribution of skin-associated microbial species. These mixes were used to estimate the resolution of two different metagenomic experiments - 16S and WGS - and to evaluate several different bioinformatics approaches for taxonomic read classification. In all test cases, WGS approaches provide much more accurate results, in terms of taxa prediction and abundance estimation, in comparison to those of 16S. Furthermore, we demonstrate that a 16S dataset, analysed using different state of the art techniques and reference databases, can produce widely different results. In light of the fact that most forensic metagenomic analysis are still performed using 16S data, our results are especially important. Show less
The current field of metagenomics can be summarised by three main questions: "Who is in metagenome?" (or "How complex the metagenome is?"), "What are they doing?" and "What is the difference... Show moreThe current field of metagenomics can be summarised by three main questions: "Who is in metagenome?" (or "How complex the metagenome is?"), "What are they doing?" and "What is the difference between two metagenomes?".This research was dedicated to creating new and evaluating already existing methods answering these questions. Show less
The use of N fertilizers has increased worldwide in the past century. While this increased input of N has increased food productivity, it has also contributed to decreases in biodiversity, soil... Show moreThe use of N fertilizers has increased worldwide in the past century. While this increased input of N has increased food productivity, it has also contributed to decreases in biodiversity, soil quality and environmental health, including increases in greenhouse gas emissions. These emissions in agricultural soils are largely carried out by the soil microbiome, or the microorganisms living in the soil and transforming N fertilizers to different forms. Here, the overall research aim was to gain detailed insight into the effects of nitrogen fertilizer schemes, including long term fertilization, on soil microbial communities. To do this, I applied next-generation sequencing technology and associated bioinformatics analyses to field experiments in the Netherlands and in Brazil. Show less
Soil biodiversity is huge and determines largely the functioning of terrestrial ecosystems both at the ‘macro’ and the ‘micro’ level. Despite the general acceptance of the large impact of land use... Show moreSoil biodiversity is huge and determines largely the functioning of terrestrial ecosystems both at the ‘macro’ and the ‘micro’ level. Despite the general acceptance of the large impact of land use and other human activities on species loss in terrestrial ecosystems, their effects on microbial species reduction and the consequences are largely unknown. A major reason is the scarcity of experimental approaches to assess the relevance of soil microbial diversity for the functioning of soil ecosystems. The main goal of the study described in this thesis was to obtain better understanding of the diversity, structuring and functioning of bacterial communities in soil and and rhizosphere. With that purpose, we initially applied the rather old dilution approach to manipulate the diversity of microbial communities in soil by inoculation and subsequent incubation of more or less diluted soil suspensions in pre-sterilized soils. Show less
