Next-generation sequencing approaches in microbiome research have allowed surveys of microbial communities, their genomes, and their functions with higher sensitivity than ever before. However, this ...sensitivity is a double-edged sword because these tools also efficiently detect contaminant DNA and cross-contamination, which can confound the interpretation of microbiome data. Therefore, there is an urgent need to integrate key controls into microbiome research to improve the integrity of microbiome studies. Here, we review how contaminant DNA and cross-contamination arise within microbiome studies and discuss their negative impacts, especially during the analysis of low microbial biomass samples. We then identify several key measures that researchers can implement to reduce the impact of contaminant DNA and cross-contamination during microbiome research. We put forward a set of minimal experimental criteria, the ‘RIDE’ checklist, to improve the validity of future low microbial biomass research.
There is increasing interest in applying metagenomic techniques to find correlations between microorganisms and disease.
Metagenomic techniques are highly sensitive and can detect contaminant DNA (DNA from sources other than the samples under study) and cross-contamination (DNA exchange between samples).
Recent studies have shown that contaminant DNA and cross-contamination can confound metagenomic studies, especially for sample types that have low microbial biomass.
There is an urgent need for the field to adopt authentication criteria to prevent future metagenomic studies from falling prey to the pitfalls of contaminant DNA and cross-contamination.
Numerous biological and cultural factors influence the microbial communities (microbiota) that inhabit the human mouth, including diet, environment, hygiene, physiology, health status, genetics, and ...lifestyle. As oral microbiota can underpin oral and systemic diseases, tracing the evolutionary history of oral microbiota and the factors that shape its origins will unlock information to mitigate disease today. Despite this, the origins of many oral microbes remain unknown, and the key factors in the past that shaped our oral microbiota are only now emerging. High throughput DNA sequencing of oral microbiota using ancient DNA and comparative anthropological methodologies has been employed to investigate oral microbiota origins, revealing a complex, rich history. Here, I review the current literature on the factors that shaped and guided oral microbiota evolution, both in Europe and globally. In Europe, oral microbiota evolution was shaped by interactions with Neandertals, the adaptation of farming, widespread integration of industrialization, and postindustrial lifestyles that emerged after World War II. Globally, evidence for a multitude of different oral microbiota histories is emerging, likely supporting dissimilarities in modern oral health across discrete human populations. I highlight how these evolutionary changes are linked to the development of modern oral diseases and discuss the remaining factors that need to be addressed to improve this embryonic field of research. I argue that understanding the evolutionary history of our oral microbiota is necessary to identify new treatment and prevention options to improve oral and systemic health in the future.
Understanding the evolutionary history of microbial communities within the human body (microbiota) will provide key insights into the origins of numerous modern‐day diseases. Ancient microbial DNA ...preserved in calcified dental plaque (calculus) now provides an opportunity to reconstruct past oral microbiota and examine how these microbes are linked with health and disease through time. We use High Throughput Sequencing to obtain ancient and modern microbial DNA from dental calculus on four continents over the past 48,000 years, reconstructing distinct oral microbiota evolutionary histories at each location. Highly resolved microbiota histories in Europe suggest that local environments, adoption of different diets, interbreeding with other hominids, and exposure to the Industrial Revolution are key drivers of microbiota shifts over time and likely underpin both oral and chronic disease origins. On other continents, oral microbial communities were distinct from those in Europe, highlighting the need to include diverse populations in evolutionary reconstructions. For example, South American oral microbiota was influenced by corn utilization and adaptation to specific environments (i.e., high altitude vs coastal) and contained distinct species linked with oral disease. As microbiota histories in each location are distinct, we theorize that they play distinct roles in the development of disease, both oral and systemic, across different populations, with profound implications for the Indigenous health gap. Together, these data provide the first glimpse of global human microbiota evolution in real‐time and provide the foundation to understand why certain bacterial communities are now linked to disease in a modern world.
Oral microbiome transplantation (OMT) is a novel concept of introducing health-associated oral microbiota into the oral cavity of a diseased patient. The premise is to reverse the state of oral ...dysbiosis, and restore the ecological balance to maintain a stable homeostasis with the host immune system. This study will assess the effectiveness, feasibility, and safety of OMT using an interdisciplinary approach.
To find donors suitable for microbial transplantation, supragingival plaque samples will be collected from 600 healthy participants. Each sample (200μL) will subsequently be examined in two ways: 1) 100μL of the sample will undergo high-throughput 16S rRNA gene amplicon sequencing and shotgun sequencing to identify the composition and characterisation of a healthy supragingival microbiome, 2) the remaining 100μL of the plaque sample will be mixed with 25% artificial saliva medium and inoculated into a specialised in-vitro flow cell model containing a hydroxyapatite disk. To obtain sufficient donor plaque, the samples would be grown for 14 days and further analysed microscopically and sequenced to examine and confirm the growth and survival of the microbiota. Samples with the healthiest microbiota would then be incorporated in a hydrogel delivery vehicle to enable transplantation of the donor oral microbiota. The third step would be to test the effectiveness of OMT in caries and periodontitis animal models for efficacy and safety for the treatment of oral diseases.
If OMTs are found to be successful, it can form a new treatment method for common oral diseases such as dental caries and periodontitis. OMTs may have the potential to modulate the oral microbiota and shift the ecological imbalances to a healthier state.
Studies of insect assemblages are suited to the simultaneous DNA‐based identification of multiple taxa known as metabarcoding. To obtain accurate estimates of diversity, metabarcoding markers ideally ...possess appropriate taxonomic coverage to avoid PCR‐amplification bias, as well as sufficient sequence divergence to resolve species. We used in silico PCR to compare the taxonomic coverage and resolution of newly designed insect metabarcodes (targeting 16S) with that of existing markers 16S and cytochrome oxidase c subunit I (COI) and then compared their efficiency in vitro. Existing metabarcoding primers amplified in silico <75% of insect species with complete mitochondrial genomes available, whereas new primers targeting 16S provided >90% coverage. Furthermore, metabarcodes targeting COI appeared to introduce taxonomic PCR‐amplification bias, typically amplifying a greater percentage of Lepidoptera and Diptera species, while failing to amplify certain orders in silico. To test whether bias predicted in silico was observed in vitro, we created an artificial DNA blend containing equal amounts of DNA from 14 species, representing 11 insect orders and one arachnid. We PCR‐amplified the blend using five primer sets, targeting either COI or 16S, with high‐throughput amplicon sequencing yielding more than 6 million reads. In vitro results typically corresponded to in silico PCR predictions, with newly designed 16S primers detecting 11 insect taxa present, thus providing equivalent or better taxonomic coverage than COI metabarcodes. Our results demonstrate that in silico PCR is a useful tool for predicting taxonomic bias in mixed template PCR and that researchers should be wary of potential bias when selecting metabarcoding markers.
•Microbes from urban green spaces may help to shape the human microbiome.•After urban green space exposure, skin and nasal microbial diversity increased.•Skin and nasal microbial composition became ...more similar to environmental sources.•Nasal microbiota are more variable between sites and individuals than skin microbiota.
In industrialized countries, non-communicable diseases have been increasing in prevalence since the middle of the 20th century. While the causal mechanisms remain poorly understood, increased population density, pollution, sedentary behavior, smoking, changes in diet, and limited outdoor exposure have all been proposed as significant contributors. Several hypotheses (e.g. Hygiene, Old Friends, and Biodiversity Hypotheses) also suggest that limited environmental microbial exposures may underpin part of this rise in non-communicable diseases. In response, the Microbiome Rewilding Hypothesis proposes that adequate environmental microbial exposures could be achieved by restoring urban green spaces and could potentially decrease the prevalence of non-communicable diseases. However, the microbial interactions between humans and their surrounding environment and the passaging of microbes between both entities remains poorly understood, especially within an urban context.
Here, we survey human skin (n = 90 swabs) and nasal (n = 90 swabs) microbiota of three subjects that were exposed to air (n = 15), soil (n = 15), and leaves (n = 15) from different urban green space environments in three different cities across different continents (Adelaide, Australia; Bournemouth, United Kingdom; New Delhi, India). Using 16S ribosomal RNA metabarcoding, we examined baseline controls (pre-exposure) of both skin (n = 16) and nasal (n = 16) swabs and tracked microbiota transfer from the environment to the human body after exposure events. Microbial richness and phylogenetic diversity increased after urban green space exposure in skin and nasal samples collected in two of the three locations. The microbial composition of skin samples also became more similar to soil microbiota after exposure, while nasal samples became more similar to air samples. Nasal samples were more variable between sites and individuals than skin samples.
We show that exposure to urban green spaces can increase skin and nasal microbial diversity and alter human microbiota composition. Our study improves our understanding of human-environmental microbial interactions and suggests that increased exposure to diverse outdoor environments may increase the microbial diversity, which could lead to positive health outcomes for non-communicable diseases.
Regular biological surveys are essential for informed management of freshwater ecosystems. However, current morphology-based biodiversity surveys can be invasive, time-consuming, and financially ...expensive. Recently, environmental DNA (eDNA) sequencing has been suggested as an alternative non-invasive, time- and cost-effective biological survey tool. However, eDNA sequencing tools require experimental validation in natural ecosystems before confidence in their use can be assumed. In this study, we compare fish community data obtained via eDNA metabarcoding to that of conventional fyke netting within two complex and drought-prone river systems. We also compare different eDNA sampling strategies and genetic markers for detecting rare and threatened fish species. We were able to detect 100% of the fyke net caught-species from eDNA when appropriate sampling strategies were used, including threatened and invasive species. Specifically, we found that two 1L water samples per site were insufficient for detecting less abundant taxa; however, five 1L samples per site enabled a 100% detection rate. Further, sampling eDNA from the water column appeared to be more effective for detecting fish communities than eDNA from sediments. However, on a per site basis, community discrepancies existed between the two methods, highlighting the benefits and limitations of both approaches. We demonstrate that careful interpretation of eDNA data is crucial as bioinformatic identification of sequences, without logical inference or local knowledge, can lead to erroneous conclusions. We discuss these discrepancies and provide recommendations for fish eDNA metabarcoding surveys.
•Fyke net and eDNA surveys of freshwater fish populations were compared.•Five 1L water samples enabled eDNA detection of 100% of fyke net-caught species.•Water eDNA more representative of fyke net-caught species than sediment eDNA•Multiple genetic markers increased detection success.•Discrepancies between the two methods existed on a per site basis.•Inappropriate marker choice and data interpretation can give rise to erroneous data.
Ancient DNA analysis of dental calculus Weyrich, Laura S.; Dobney, Keith; Cooper, Alan
Journal of human evolution,
February 2015, 2015-Feb, 2015-02-00, 20150201, Volume:
79
Journal Article
Peer reviewed
Dental calculus (calcified tartar or plaque) is today widespread on modern human teeth around the world. A combination of soft starchy foods, changing acidity of the oral environment, genetic ...pre-disposition, and the absence of dental hygiene all lead to the build-up of microorganisms and food debris on the tooth crown, which eventually calcifies through a complex process of mineralisation. Millions of oral microbes are trapped and preserved within this mineralised matrix, including pathogens associated with the oral cavity and airways, masticated food debris, and other types of extraneous particles that enter the mouth. As a result, archaeologists and anthropologists are increasingly using ancient human dental calculus to explore broad aspects of past human diet and health. Most recently, high-throughput DNA sequencing of ancient dental calculus has provided valuable insights into the evolution of the oral microbiome and shed new light on the impacts of some of the major biocultural transitions on human health throughout history and prehistory. Here, we provide a brief historical overview of archaeological dental calculus research, and discuss the current approaches to ancient DNA sampling and sequencing. Novel applications of ancient DNA from dental calculus are discussed, highlighting the considerable scope of this new research field for evolutionary biology and modern medicine.
Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and ...reagents is especially problematic when examining samples with low endogenous content (e.g., skin swabs, tissue biopsies, ice, water, degraded forensic samples or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high‐throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no‐template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over 5 years to characterize long‐term contaminant diversity. We additionally compared the contaminant content within a home‐made silica‐based extraction method, commonly used to analyse low endogenous content samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to modern molecular biology laboratories, and changed over time according to researcher, month and season. The commercial kit also contained higher microbial diversity and several human‐associated taxa in comparison to the home‐made silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low‐biomass metagenomic studies: (a) extraction blank controls should be included and sequenced with every batch of extractions and (b) the contributions of laboratory contamination should be assessed and reported in each high‐throughput metagenomic study.
While microbial communities in the human body (microbiota) are now commonly associated with health and disease in industrialised populations, we know very little about how these communities ...co-evolved and changed with humans throughout history and deep prehistory. We can now examine these communities by sequencing ancient DNA preserved within calcified dental plaque (calculus), providing insights into the origins of disease and their links to human history. Here, we examine ancient DNA preserved within dental calculus samples and their associations with two major cultural periods in Japan: the Jomon period hunter–gatherers approximately 3000 years before present (BP) and the Edo period agriculturalists 400–150 BP. We investigate how human oral microbiomes have changed in Japan through time and explore the presence of microorganisms associated with oral diseases (e.g. periodontal disease, dental caries) in ancient Japanese populations. Finally, we explore oral microbial strain diversity and its potential links to ancient demography in ancient Japan by performing phylogenomic analysis of a widely conserved oral species—
Anaerolineaceae
oral taxon 439. This research represents, to our knowledge, the first study of ancient oral microbiomes from Japan and demonstrates that the analysis of ancient dental calculus can provide key information about the origin of non-infectious disease and its deep roots with human demography.
This article is part of the theme issue ‘Insights into health and disease from ancient biomolecules’.
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