Welcome back to the fascinating world of human skin microbiota!
In this second part of our series, we will take a closer look into the topic and explore a powerful approach used to analyze the composition and diversity of skin microbiota.
As we discussed in the previous post, microbiome-friendly skincare products have gained significant momentum, aiming to protect and strengthen our skin as the first line of defense in our skincare routine. Maintaining a balance and diverse skin microbiota is crucial for the proper functioning of this protective shield. By promoting the growth of beneficial microorganisms and preventing the colonization of pathogenic ones, we can achieve healthier, more radiant, and aesthetically pleasing skin.
However, a fundamental question arises: How can we accurately evaluate the composition and diversity of the human skin microbiome??? This is where METAGENOMICS comes into play and provides us with valuable insights.
Microbiological culture and Metagenomics
Whenever we think about studying microbiology, our immediate thoughts gravitate toward surveying, culturing, staining, identifying, and manipulating microbes. However, when dealing with samples containing thousands of microbial species, this traditional culture-based method falls short, and this is where metagenomics steps in and revolutionizes the game!
In simple terms, culture-dependent methods are used in the lab to grow specific organisms by plating samples on specialized media that encourage their growth. These organisms are then identified based on characteristics such as colony appearance, growth patterns on different media, and metabolic activities observed through biochemical tests. On the other hand, metagenomics is an independent-culture method that allows us to analyze the composition and diversity of the skin microbiome through direct DNA sequencing without the need for cultivation.
The question is, how and why is metagenomics utilized in skin microbiome studies? It is crucial to acknowledge that the vast majority of microbial species have never been successfully cultured in the laboratory. The limitations of laboratory conditions, including the inability to provide the necessary metabolites or signaling molecules produced by other microorganisms in the original community, hinder the successful culturing of these microorganisms. With metagenomics, genetic materials of all microbes in samples can be directly analyzed without the need for cultivation, allowing the detection of genes/microbes that may not have been previously identified by culture-based methods. This method provides a comprehensive understanding of the microbial community present on the skin, including both known and unknown microorganisms. Additionally, the culture-dependent method focuses on studying individual microorganisms in a laboratory setting, limiting the evaluation of the overall complexity and diversity of a microbial community. In contrast, metagenomics allows for the sequencing of all DNA in a sample, enabling real-time assessment of the microbial composition of a community. It provides insights into the diversity of the microbial community over time and allows investigation of their metabolic processes and interactions within the community. Moreover, because metagenomics can analyze the DNA of all microbes in samples, it allows us to detect potential pathogens or imbalances within the skin microbiome. By comparing metagenomic data from healthy and diseased skin samples, we can identify specific microbial signatures associated with certain skin conditions. This understanding opens up possibilities for targeted interventions and personalized approaches to restore balance and promote skin health.
Although metagenomics has revolutionized our understanding of microbial communities, culture-independent approaches cannot completely replace the traditional microbiological culture methods. Both methods have their strengths and limitations, and they are often used in combination to obtain a comprehensive understanding of the microbial world.
DIFFERENT APPROACHES IN METAGENOMICS
AND HOW THEY ARE DONE
Results from metagenomics analysis tell us two features of a microbial sample: microbiome structure or taxonomic diversity to define the composition of microorganisms present, and microbiome functions to see the biological roles that microbes of the community perform.
There are two main approaches in metagenomics analysis, which are SHOTGUN METAGENOMICS and AMPLICON METAGENOMICS/METABARCODING.
The major and most important difference between these two approaches lies in their scope of analysis. Shotgun metagenomics focuses on sequencing the entire genetic materials (DNA or RNA) present in samples, including microbial and host DNA, providing a comprehensive view of the entire microbial community and allowing for the discovery of novel species and functional genes. As a result, it provides insights into the metabolic potential, functional diversity, and interactions within the microbiome.
However, amplicon metagenomics involves amplifying and sequencing target regions of the microbial DNA, typically using genetic markers such as the 16S rRNA gene (bacteria), ITS region (fungi), chloroplast markers (plants), and so on. Thus, it just provides information about the taxonomic composition of the microbial community but does not provide extensive information on functional genes.
Before any further discussion about the differences between these two approaches, let me introduce you to the typical procedure in metagenomics studies of the human skin microbiome. Research always starts with experiment design to
ensure scientific rigor. The next step involves sample collection from the human skin, which contains a mixture of genetic material from both the microorganisms inhabiting the skin and also the host. DNA extraction is then performed to isolate and purify the microbial DNA for the next stages. Subsequently, the extracted DNA undergoes library preparation, where it is processed to create sequencing libraries. This crucial step precedes sequencing, which involves generating sequence data from the libraries. Finally, the collected data is analyzed using bioinformatic tools and pipelines, allowing for taxonomic and functional profiling through data annotation.
Step 1: Experiment design
To ensure the coherence and logic of our experimental design, it is essential to have a clear aim for the project. The aim sets the direction and purpose of the research, guiding the design of the experiment and enabling it to address specific questions. It also helps us to avoid any ambiguity and/or confusion in the experimental design, ensuring that research questions are effectively addressed.
There are two controls that need to be included in a metagenomics study. The first one is the negative control utilized for mapping to the environment/host genomes in the sequencing process. The role of negative control is to ensure that there is no contamination from the laboratory, environment, and chemicals used. By sequencing and analyzing these negative controls, it can be determined whether or not any DNA not present in the initial test sample is introduced during sample collection, processing, or analysis. The second one is the positive controls, which are mock communities containing known ratios of microbial species or strains and can be sequenced alongside the test samples. Because we can compare the expected ratios to the observed ratios, the accuracy and efficiency of various steps in the process, such as amplification and library preparation, can be assessed.
Step 2: Sample collection
Skin microbiome samples are typically collected using cotton buds or swabs and stored in appropriate containers. However, a challenge in this process is how to minimize human DNA contamination and ensure a high bacterial DNA recovery. To address this issue, swabbing is considered the optimal method for sample collection, as it tends to have the lowest human DNA contamination (less than 3%). This reduced contamination is likely due to the DNA degradation that occurs after cell enucleation, which is a consequence of squamous formation. Additionally, studies have shown that the taxonomic structure of skin bacteria communities obtained through methods such as scratching or biopsy, which penetrate deeper into the subsurface of the skin, were not found to be statistically different from those based on swabbing. Thus, by utilizing the swabbing technique, researchers can obtain skin microbiome samples with minimal interference from human DNA, ensuring the focus remains on analyzing the microbial composition accurately.
Step 3: DNA/RNA extraction
The collected samples undergo DNA extraction to isolate and purify the microbial DNA. Various techniques are employed to break open the microbial cells, separate the DNA from other cellular components and obtain the purified genetic materials in a more concentrated volume.
Step 4: Library preparation
In shotgun metagenomics, the extracted DNA is fragmented into smaller pieces by different methods including sonication (applying high-frequency sound waves) or enzyme digestion. Specific DNA adapters are subsequently ligated to the ends of DNA fragments before being sequenced.
In amplicon or metabarcoding, specific target regions (also called genetic markers) are selected for amplification which is commonly used for microbial community analysis, such as the 16S rRNA gene for bacterial studies (from V1 to V9) or the ITS region for fungal studies. Unlike bacteria and archaea, viruses do not possess universally conserved DNA markers, therefore, the shotgun approach is the only available option to study the viral community within a sample. Subsequently, PCR is performed using primers that specifically target the selected regions which are designed to bind to conserved regions flanking the target one, allowing for selective amplification of the desired DNA target. Similarly to shotgun metagenomics, adapters containing sequencing-specific sequences are ligated to the amplicons generated from the PCR step, enabling subsequent steps in sequencing.
Step 5: DNA sequencing
In this step, high-throughput sequencing technologies, such as next-generation sequencing (NGS) would be used, generating massive amounts of sequence data, representing the genetic content of the microbial community within human skin samples.
In shotgun metagenomics, the resulting DNA library, consisting of DNA fragments from the entire genome of microorganisms present in the sample, is then subjected to sequencing. During sequencing, the DNA fragments are amplified and the sequences of the individual fragments are determined.
In amplicon metagenomics, the amplicon library, consisting of target regions amplified from the microbial community, is subjected to sequencing to determine the sequence of the desired regions. However, one limitation is that in certain cases, the detection may be challenging when only a single variable region is analyzed. To address this issue, an approach is to combine the sequencing of multiple variable regions simultaneously. By sequencing multiple variable regions, the ability to identify microorganisms as well as assess the composition and diversity in samples can be improved. This approach allows for a more comprehensive understanding of the microbial community present in the sample.
Step 6: Data analysis
After getting the results of the DNA sequence, bioinformatics would solve all the remaining questions.
Besides, to eliminate human DNA contamination during bioinformatic analysis, the sequencing data can be aligned with a reference human DNA library. This allows for the removal or exclusion of sequences that originate from the human genome, ensuring that the analysis focuses specifically on microbial DNA.
The data analysis process for metagenomics shotgun and amplicon approaches differs due to their distinct sequencing strategies and target regions.
Taxonomic Profiling: Shotgun metagenomics allows for comprehensive taxonomic profiling of the entire microbial community. It involves aligning sequenced reads to reference databases, such as the NCBI's non-redundant nucleotide database, to identify and classify the microorganisms present. Amplicon metagenomics focuses on targeted regions, making it more suitable for higher-resolution taxonomic profiling of specific groups, such as bacteria or fungi.
To evaluate the diversity and compositions of microorganisms in microbial samples, here are some key values:
Alpha diversity: Alpha diversity refers to the diversity within a single sample or microbial community. It measures the richness (number of different species or taxa) and evenness (relative abundance of different species or taxa) within the community. Alpha diversity metrics provide insights into the overall diversity and distribution of species within a specific sample.
Beta diversity: Beta diversity measures the dissimilarity or differentiation between different samples or microbial communities. It compares the species composition and abundance across samples to assess how communities vary in their microbial composition. Beta diversity metrics provide insights into the differences and similarities in microbial communities between samples or conditions.
OTUs (Operational Taxonomic Units): OTUs are clusters of sequences that are grouped together based on a pre-defined similarity threshold. OTUs are used as a proxy for taxonomic units and are commonly employed in amplicon-based metagenomics studies. Each OTU represents a distinct taxonomic group, although it may not necessarily correspond to a specific species. OTUs help identify and quantify the diversity and abundance of microbial taxa in a sample.
These values are assessed and calculated using various statistical and bioinformatics methods. They provide important information about the microbial community structure, diversity, and potential functional capabilities.
Functional Annotation: Shotgun metagenomics provides insights into the functional potential of the microbial community. By comparing the sequenced reads to reference databases, functional annotations can be assigned to identify the genes and metabolic pathways present. Amplicon metagenomics is limited in the functional analysis since it primarily focuses on community composition rather than the full gene content.
In conclusion, metagenomics is a powerful sequencing technology that offers a comprehensive understanding of the skin microbiome. It allows researchers to explore how the microbiome interacts with its host, how it responds to different skin conditions, and how it contributes to both the taxonomic and functional aspects of the skin microbial community. This knowledge opens up possibilities for new innovations in skincare, including the development of active ingredients and formulas that promote aesthetically healthy and radiant skin. By harnessing the potential of metagenomics, we can pave the way for personalized approaches to skincare and improve our overall understanding of the intricate relationship between the skin and its microbial inhabitants.
Chloe Vo || Cosmetics x Science
References:
8/ https://www.everydayhealth.com/skin-beauty/everything-you-need-to-know-about-the-skin-microbiome/
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