Microbiome innovations: next-generation sequencing for microbial profiling.

The human microbiome comprises all bacteria, fungi and viruses that inhabit our bodies, including their genetic material. While humans have around 24,000 genes in their genome, it is estimated that the genetic material from bacteria living symbiotically with us contain more than 360 times more [1]. This genetic material can aid research by providing insights into which microbes are present in an individual – as part of a process known as microbial profiling.

Although the sheer size of the microbiome and its genetic material presents a significant analytical challenge – and the first microbial profiling methods to emerge came with high costs and slow speeds – advances in sequencing technologies such as DNA and genome sequencing, metagenomics and transcriptomics, which can profile composition as well as functional aspects of microbial communities, are making it easier, faster and cheaper. These advances have revolutionized the study of microbial communities, with the technologies available making it possible to extract valuable personalized information from a sample. This can help us understand the importance of the microbiome for our wellbeing and how our microbial composition changes in relation to factors such as age and disease.

Microbial profiling primarily evolved for use to study the gut microbiome – one of the most extensively characterized microbial communities in humans with known roles in human health, such as immune system regulation and aiding digestion. However, more recently, the skin microbiome and its importance has gained increasing attention. Here, microbial profiling insights can help us link skin microbial compositional changes to skin health and disease, and the insights can also be used to support the development of diagnostic tools, new therapeutics and dermatological and cosmetic skincare products.

In this article, we will explore the evolution and capabilities of microbial profiling with particular focus on its application to the skin microbiome, and illustrate how the technology can be used within the personal care space to help understand and support human health. 

Next-generation sequencing for microbial profiling  

Microbial communities were traditionally explored using culture-based methods, where microorganisms are grown on media in the laboratory to isolate and identify individual species. However, as this approach carries a bias for microorganisms that thrive in artificial growth conditions, it underestimates the total diversity of a microbial community. This also led to certain microbial species – such as skin fungi like Malassezia – being missed in earlier profiling studies (as Malassezia require special growth conditions once isolated from the natural habitat of the skin). More recently, culture-independent methods such as DNA sequencing have become increasingly popular as these approaches can escape the bias imposed by culture and to capture the complete diversity of the microbiome [2,3,4].

DNA sequencing is a broad term that refers to any method used to determine the nucleotide sequence of a DNA molecule. In the microbiome world, the original DNA sequencing approaches utilized sequence variation in conserved taxonomic markers as molecular fingerprints to identify members of microbial communities. Continued advances have led to the evolution of next-generation sequencing – a specific type of DNA sequencing that uses high-throughput technologies to rapidly sequence millions of DNA or RNA fragments in parallel, generating large amounts of data in a relatively short amount of time. These technologies made sequencing easier and more affordable – increasing access for researchers. There are now various types of next-generation sequencing, broadly classed under whole-genome sequencing, transcriptome sequencing and metagenomic sequencing [2,5].

Two next-generation sequencing techniques often used to study a microbial community include: (1) amplicon sequencing, a highly targeted approach that enables researchers to analyze genetic variation in specific genomic regions via deep sequencing of PCR products (amplicons); and (2) shotgun metagenomic sequencing studies, which allow researchers to comprehensively sample all genes in all organisms present in a given complex sample [2,3,5].

In amplicon sequencing, primers are used to amplify conserved regions within a kingdom for microbial community analysis. For bacteria, the 16S rRNA region of the ribosomal gene, which is commonly used as a marker gene for bacterial identification and classification, is amplified; whereas for fungi, the internal transcribed spacer 1 (ITS1) region is targeted. While this method has some strengths, such as its cost-effectiveness, it is usually limited to genus-level resolution [2,4,6].

By contrast, shotgun metagenomics, which is increasingly used in both gut and skin microbiome profiling, captures the entire complement of genetic material in a sample (including human, bacterial, fungal, viral and archaeal) without a targeted amplification step and does not sequence specific target regions. This allows for unbiased taxonomic identification at species and even strain level, and for relative kingdom abundances to be inferred. This high resolution is relevant when strains within a species harbour different gene content that determines functional differences – such as in the case of Cutibacterium acnes or Staphylococcus aureus on the skin [2,4,7,8]. However, this increased complexity also comes with higher sequencing costs and more complex data analysis [4,9].

Current state of the technology

DNA and biomass

The use of shotgun metabolomics for skin samples, which have a low microbial biomass, is challenging as it requires a high starting amount of DNA [4,10,11]. In addition, skin samples contain high levels of host DNA, where it can represent more than 90% of the total extracted DNA. Therefore, although host DNA sequences can be removed later during data processing, a large part of the reads have already been spent on sequencing the human genome. This results in increased costs due to the deep sequencing runs required to yield relevant information [4,12].

Absolute quantification

As next-generation sequencing instruments have a fixed capacity, the data obtained for all detected taxa will be described as relative abundances – which are mutually dependent – but not as absolute numbers. This poses a challenge, as relative abundances can lead to misleading results [4,13]. In addition, the quantification of skin microorganisms is important to address the bioburden of common microbes and whether it increases in specific disorders. Different methods have been integrated into next-generation sequencing pipelines to allow for quantitative data. However, they increase the cost and complexity of the data generated and are not yet widely used in the human microbiome field [4,13,14].

Analytical challenges

The next-generation sequencing revolution presents new analytical challenges, due in part to the complexity and size of the resultant datasets. It is a complex and multistep preparation procedure, with a variety of potential adaptations which can all result in measurement bias. Next-generation sequencing experiments are also expensive, which can limit experimental replication. These factors, plus the lack of suitable reference materials, present challenges for the standardization of metagenomic measurements and the comparison of experimental results [2,4].

Despite the challenges, a number of companies and research institutions have leapt to the challenge of continuing to advance next-generation sequencing technology approaches and overcome the remaining issues while also reducing costs – further broadening and improving the potential research and clinical pathways where microbial profiling can support. 

Bio-Me, for example, is transforming microbiome analysis by providing a skin microbiome precision profiling platform that makes it possible to analyze hundreds of samples in less than a day, with high resolution and reproducibility. Combining an exclusive biobank, an extensive patient database and a panel of key bacterial and fungal targets for both the gut and skin, the platform uses a DNA-sequencing approach known as quantitative polymerase chain reaction (qPCR) to deliver accurate information about the microbiome in a cost-effective and high-throughput format.

The use of qPCR to detect, characterize and quantify the nucleic acids that make up DNA offers high-level resolution equivalent to that of shotgun metagenomics, down to species and subspecies level, and is not impacted by host DNA. This information can be used to provide key insights into an individual’s microbiome and any disruptions, and to support clinical testing or product development – for example, to investigate effect of a biotic treatment or cream application. 

Applying microbial profiling – opportunities in personal care  

Microbial profiling is a powerful tool that can help us understand the complex interactions between the microbiome, genetics and the exposome – by providing information about the composition and function of the microbial communities that inhabit the human body, as well as how they are influenced by factors such as host genetics and the environment (find out more about these interactions here). By gaining deeper insights and building a more integrated framework in this way, we can gain a better understanding of how the skin microbiome can influence human health and how its dynamics feed into a range of different areas – for example:

Skin disease and treatment:

• Working from an established baseline, microbial profiling can now provide insights into the composition and activity of microbial communities in the human body, allowing for the identification of potential pathogens or dysbiosis that may contribute to disease or even targets for novel therapies [6]. For the skin specifically, microbial profiling can help identify the presence of pathogenic microorganisms or dysbiosis that may contribute to various skin disorders, such as acne, eczema and psoriasis [15]. These insights can also be used to develop new diagnostic tools and treatments that target the skin microbiome.

Skin aging:

• Microbial profiling can help understand the changes that occur in the skin microbiome with age. This can provide insights into the aging process and the development of age-related skin conditions, as microbiome composition dynamically changes throughout the human lifespan and has a bidirectional impact on health and illnesses, and guide new approaches for targeted microbial therapy for skin aging [16,17].

Cosmetics and skincare product development:

• Microbial profiling can be used to evaluate the effectiveness of cosmetics and skincare products, as well as any potential harm caused by the products, by assessing their impact on the skin microbiome. This can help develop new products that promote skin health by supporting the growth of beneficial microorganisms and reducing the growth of harmful ones [18,19] (see also DSM’s approach to microbiome-friendly skincare; and an interview on developing microbiome-friendly products).

Biotics and live biotherapeutics:

• By helping to identify beneficial microorganisms and their functional properties, microbial profiling can also be used to help develop ingestible biotics and live biotherapeutics that can help promote human health by improving gut microbiome composition, immune function and overall wellbeing. Such information will facilitate the expansion of knowledge to the development of topical pre- pro- and postbiotics and live biotherapeutic products for different skin conditions [2,19].

As technology continues to advance and profiling studies continue to dive deeper into microbial profiling for human health and disease, we can expect to gain many more insights in the near future. These insights can help drive a deeper understanding of host–microbial dynamics, more precise diagnostic and prognostic processes and more personalized treatments and products – both for the gut and skin.

References

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