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Quick overview of the microbial ecology analyses using bioinformatics tools. The figure shows the succession of analyses from alpha diversity to differential abundance and the three stages of analysis: data transformation, visualization tools, statistical analysis, of each step.
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High-throughput DNA sequencing (HTS) has changed our understanding of the microbial composition present in a wide range of environments. Applying HTS methods to air samples from different environments allows the identification and quantification (relative abundance) of the microorganisms present and gives a better understanding of human exposure to...
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... offer a guideline on how to build a metadata mapping file: http://qiime.org/scripts/validate_mapping_file.html. Figure 1 is a quick overview showing the succession of all the major steps of the microbial ecology analyses using bioinformatics tools that will be discussed in this work. Each step is divided into three stages: data transformation, visualisation and statistical analysis. ...
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... • Bioinformatics tools and software packages are used to analyze sequencing data, construct phylogenetic trees, and assess microbial diversity and community composition [46]. ...
Strengthening agriculture is crucial for ensuring food security for the world’s expanding population. Endophytes, microorganisms that live within plant tissues without harming the host, can colonize these tissues. They promote plant growth, serve as biocontrol agents, and naturally shield the host from a variety of biotic and abiotic challenges. Each year, the emergence of numerous stresses in crops leads to a decrease in yield, resulting in significant losses. To mitigate these losses and manage plant pests and diseases, various effective strategies should be employed. Endophytes, being environmentally friendly, non-toxic, and cost-effective, could serve as an alternative for farmers aiming for sustainable agriculture. This chapter will discuss the biochemical, molecular, and genetic mechanisms of endophytes in enhancing stress tolerance in different crops and their role as biological control agents. Furthermore, endophytes and their metabolites should be explored as potential beneficial agents in the biological control of plant pests and diseases, extending the investigation up to the multi-omics level.
... While Chiarello et al. [23] found differences in microbial communities analyzed with the OTU vs. the ASV approach, other studies did not highlight significative differences in sample compositions based on the approach [19,21,24]. Overall advantages and disadvantages have been highlighted by using both approaches [11,14,16]. In regards to studies analyzing the species compositions of animal foodstuffs, no significative differences related to the usage of ASV-based BPs or OTU-based BPs were factually highlighted [25,27]. ...
... It is often considered an ideal first programming language due to its easy learning curve for beginners [39]. Therefore, it is even more important for those interested in using a bioinformatics tool such as DADA2 [16] to be familiar with it. Indeed, in the field of bioinformatics, Python and R have emerged as the dominant programming languages, and R, in particular, has a strong and potentially long-lasting position within bioinformatics [39]. ...
Next Generation Sequencing Technologies (NGS), particularly metabarcoding, are valuable tools for authenticating foodstuffs and detecting eventual fraudulent practices such as species substitution. This technique, mostly used for the analysis of prokaryotes in several environments (including food), is in fact increasingly applied to identify eukaryotes (e.g., fish, mammals, avian, etc.) in multispecies food products. Besides the “wet-lab” procedures (e.g., DNA extraction, PCR, amplicon purification, etc.), the metabarcoding workflow includes a final “dry-lab” phase in which sequencing data are analyzed using a bioinformatic pipeline (BP). BPs play a crucial role in the accuracy, reliability, and interpretability of the metabarcoding results. Choosing the most suitable BP for the analysis of metabarcoding data could be challenging because it might require greater informatics skills than those needed in standard molecular analysis. To date, studies comparing BPs for metabarcoding data analysis in foodstuff authentication are scarce. In this study, we compared the data obtained from two previous studies in which fish burgers and insect-based products were authenticated using a customizable, ASV-based, and command-line interface BP (BP1) by analyzing the same data with a customizable but OTU-based and graphical user interface BP (BP2). The final sample compositions were compared statistically. No significant difference in sample compositions was highlighted by applying BP1 and BP2. However, BP1 was considered as more user-friendly than BP2 with respect to data analysis streamlining, cost of analysis, and computational time consumption. This study can provide useful information for researchers approaching the bioinformatic analysis of metabarcoding data for the first time. In the field of food authentication, an effective and efficient use of BPs could be especially useful in the context of official controls performed by the Competent Authorities and companies’ self-control in order to detect species substitution and counterfeit frauds.
... The developments of massive parallel sequencing (MPS) techniques allow a more profound knowledge of the microorganisms, either unraveling the entire microbial composition retrieved from a given environment both in terms of identification and quantification (relative abundance) [48] or determining the complete genome sequence of a single microorganism, aiming for a complete portrait of the gene present mainly considering aspects such as metabolic profile, virulence, antifungal resistance, or recombination [49]. ...
Fungi are amongst the most abundant and diverse organisms. Despite being widely known for their adverse role in food spoilage or as pathogens for humans, animals, or plants, they also present several beneficial effects. Fungi contribute to human well-being due to their role as decomposers, degrading decay matter into smaller molecules which can be easily used by other ecosystem members. These organisms can produce medicinal compounds or modulate protective immune responses in human intestine. Fungi intervene in diverse food processes or act as a food supply. Due to fungal diversity, the unequivocal identification of these organisms is crucial to increasing their practical applications and decreasing their adverse effects. The process of identification could be achieved through the integral sequencing of fungi genomes. However, this procedure would be time-consuming and rather cost-inefficient. Therefore, several molecular markers have been developed to overcome these limitations. The chronology of DNA-based molecular markers development can be divided into three main steps: (1) prior to the development of the PCR technique (RFLP); (2) after the development of the PCR technique (RAPD, AFLP, ISSR, VNTR, SNP, InDels, and DNA barcoding); (3) after the development of the massive parallel sequencing technique (Metabarcoding and WGS). Therefore, the present review covers an overview of the most recently developed molecular markers used for fungal detection and identification.
... The Jaccard index also showed that dogs consuming wet vs. dry foods clustered separately in SUP samples, but not in SUB samples (Figure 3). The Bray-Curtis index, which takes into account the abundance of each bacterial taxa present and estimates the dissimilarity of samples (Mbareche et al., 2020), showed that sample types (SUB, SUP) clustered separately from one another (Supplementary Figure 2). The Bray-Curtis index also showed that dogs consuming wet vs. dry foods clustered separately in SUP samples, but not in SUB samples ( Figure 3). ...
Oral microbiota play a prominent role in canine periodontal disease and wet foods are often blamed for poor oral health, but canine oral microbial communities have been poorly studied. We aimed to determine differences in oral health measures, breath odor, and oral microbiota populations of dogs fed wet or dry food. Twelve adult dogs fed either a commercial dry (extruded) or commercial wet (canned) food for 6 wk were studied. Breath samples were measured for sulfur compounds, teeth were scored for plaque, calculus, and gingivitis by a blinded veterinary dentist, salivary pH was measured, and supragingival (SUP) and subgingival (SUB) plaque samples were collected for microbiota analysis. Plaque DNA was extracted and Illumina sequencing conducted. Phylogenetic data were analyzed using the CosmosID bioinformatics platform and SAS 9.4, with P<0.05 being significant and P<0.10 being trends. Plaque coverage tended to be higher (P<0.10) in dogs fed wet vs. dry food, but other oral health scores were not different. Dogs fed dry food had higher (P<0.05) salivary pH and lower (P<0.05) breath sulfur concentrations than those consuming wet food. Bacterial alpha diversity was higher in SUP than SUB samples, and a clear separation in beta diversity was observed between sample sites on principal coordinates analysis (PCoA) plots. In SUP samples, dogs fed wet food had a higher alpha diversity than dogs fed dry food, with PCoA plots showing a separation between wet and dry food. Relative abundances of Firmicutes, Synergistetes, and 10 bacterial genera were different (P<0.05) in SUB samples of dogs fed wet vs. dry food. Relative abundances of Fusobacteria and over 20 bacterial genera were different (P<0.05) in SUP samples of dogs fed wet vs. dry food. In general, oral health-associated bacterial taxa (Pasteurella, Capnocytophaga, Corynebacterium) were higher, while bacteria associated with poor oral health (Fretibacterium fastidiosum, Filifactor alocis, Treponema medium, Tannerella forsythia, Porphyromonas canoris, Porphyromonas gingivalis) were lower in dogs fed dry food. Such shifts in the oral microbiota may impact periodontal disease risk, but longer dietary intervention studies are required to confirm their role in the disease process. Our results suggest that dogs fed dry extruded foods have lower breath odor and tooth plaque buildup and an oral microbiota population more closely associated with oral health than dogs fed wet canned foods.
... Whole genome assembly, however, would have been a useful addition to all three studies for characterising and identifying functional genes, such as for nitrification or ammonia oxidation, or for enzymes capable of breaking down recalcitrant matter.Data normalisation methods were employed for all three studies, to account for uneven sample sequencing depth. In this case, samples represent a fraction of the microbial community and the observed sequences are relative abundances.Whilst normalisation measures create even sample sizes and clarity of diversity patterns, they can introduce biases, such as the loss of reads which reduces any differences in diversity measures between samples(Mbarache et al., 2020). A PERMANOVA mixed-effect model was employed in all studies, which consisted of multiple factors. ...
This thesis focuses on the spatial variability of benthic abyssal microbes, which have important implications for biogeochemical cycling in the deep ocean. Abyssal ecosystems, considered to be between 3,000 to 6,000 m water depth, cover vast areas of the Earth’s surface. The majority of the abyssal seafloor is characterised by fine sediments, and these benthic habitats contain vast numbers of microbes with huge metabolic diversity. Abyssal microbes drive biogeochemical cycles, regulate fluxes of energy and contribute to organic carbon production and remineralization. So far, detailed assessments of the spatial distributions of benthic microbial communities in abyssal regions are still incomplete, and the influence of seafloor topography and heterogeneity on microbial distributions across a range of scales are poorly understood. Therefore, the aim of this thesis is to characterise the spatial variability of benthic microbes, which is essential for understanding their roles in benthic environments and for conducting baseline assessments of areas of the seabed that might be targeted by commercial mining activities. The spatial variation of benthic microbes in the Atlantic and Pacific oceans is the subject of three chapters, one of which focuses on the spatial variation of microbes associated with sediment and polymetallic nodules in the Clarion-Clipperton Fracture Zone (CCFZ) of the Pacific Ocean. The following chapter compares diversity and distribution of sediment microbes across three sites with varying productivity regimes in the Atlantic and Pacific. The final chapter examines the gut microbiome of holothurians, a dominant megafaunal group in the North-East Atlantic. This thesis showed that ammonia-oxidising Archaea (Thaumarchaeota) and nitrogen-cycling microbes were highly abundant in oligotrophic sediments and highlighted that these microbial taxa exhibited a spatial variability across small to large geographic scales, indicative of nîche diversity and resource partitioning. Given that chemolithoautotrophic taxa, such as ammoniaoxidisers and nitrifying microbes may form a significant source of organic carbon through inorganic carbon fixation and as microbes in general form the base of the marine food web, changes to microbial assemblages will likely have cascading impacts to higher trophic levels. Understanding the diversity patterns and functional roles of benthic microbes is vital for the sustainable use and preservation of ecosystems ahead of potential seabed exploration, exploitation and disturbance. Detailed observations are therefore necessary for modelling microbially mediated biogeochemical functions, monitoring biodiversity and ecological assessments for the preservation of ecosystem functioning.
... The relative abundance of a specific taxon was normalized and calculated as the Number of Reads for a taxon divided by the Number of Reads in Total 16S rRNA gene. The differences of bacterial abundances at both phylum and genus (the top 32 most abundant genera) levels between groups were determined by implementing the function 'Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC)' using ANCOM package in R software [23]. The randomly sub-sampled taxonomic sequences (rarefaction depth = 13,800 sequences) were applied to calculate alpha and beta diversities. ...
... Various alpha diversity indexes including bergerparker, invsimpson, npshannon, and qstat measuring phylogenic diversity, ace, bootstrap, chao, and sobs assessing microbial richness, and heip, shannoneven, simpsoneven, and smithwilson evaluating microbial evenness, in soil bacterial microbiome were assessed through Summary. Single tool in mothur and compared between groups by one-way analysis of variance (ANOVA) using vegan package in R software [23]. Beta diversity was calculated based on the calculator the tayc/jclass and Phylip distance matrix using Dist.shared ...
As a traditional agricultural system, integrated crop-livestock farms (ICLFs) involve the production of animals and crops in a shared environment. The ICLFs in the mid-Atlantic region of the United States practice sustainable manure aging or composting processes to provide an on-farm source of soil amendment for use as natural fertilizer and soil conditioner for crop production. However, crop fertilization by soil incorporation of aged manure or compost may introduce different microbes and alter the soil microbial community. The aim of this study was to characterize the influence of aged or composted manure application on the diversity of soil bacterial community in ICLFs. Soil samples from six ICLFs in Maryland were collected before (pre-crop) and during the season (2020–2021) and used to analyze soil bacterial microbiome by 16S rDNA sequencing. Results showed that both phylum- and genus-level alterations of soil bacterial communities were associated with amendment of aged or composted manure. Particularly, Proteobacteria and Actinobacteria were enriched, while Acidobacteria, Bacteroidetes, Planctomycetes, Firmicutes, and Chloroflexi were reduced after manure product application. Meanwhile, the relative abundance of Bacillus was decreased, while two zoonotic pathogens, Salmonella and Listeria, were enriched by manure amendments. Overall, animal manure amendment of soil increased the phylogenetic diversity, but reduced the richness and evenness of the soil bacterial communities. Although manure composting management in ICLFs benefits agricultural sustainable production, the amendments altered the soil bacterial communities and were associated with the finding of two major zoonotic bacterial pathogens, which raises the possibility of their potential transfer to fresh horticultural produce crops that may be produced on the manured soils and then subsequently consumed without cooking.
