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How to Design a Skin Microbiome Study, Part II: Amplicon Sequencing

In this post, the second of a 2-part series on skin microbiome research, we will discuss technical issues surrounding sequencing of human skin microbes. Read the first blog post here.

At this point, the microbial ecologist conducting a skin microbiome study has now collected all the skin samples she needs, and the DNA has been extracted. We turn to the question of how to decide on the sequencing strategy. Metagenome shotgun sequencing, in which the entire community of microbes is sequenced in an untargeted manner, can provide invaluable information about the functional potential of the microbiome, but – despite continually dropping sequencing costs – it is still expensive. The researcher in this case settles for 16S marker gene sequencing, which targets a specific region of the gene. Now, which primer pair should she choose?

Credit: stevanovicigor. Thinkstock image used with permission.

The current dogma in the field is that primers targeting regions V1-V3 are better at describing skin bacterial communities than the V4 region primer pair. (The V4 region is commonly used for studying gut communities and other environments.) This is because V1-V3-sequenced communities better recapitulate the taxonomic composition and relative abundance of “mock community” controls (Meisel et al., 2016). And V4 primers poorly amplify typical skin microbes, notably Propionibacterium and some Staphylococcus species (Meisel et al., 2016). But should V4 be discarded in favor of V1-V3?

The reason behind the V4 region’s underestimation of Propionibacterium is a single mismatch at the end of the primer that prevents efficient binding to a specific group of bacteria. To evaluate if V4 region may be a suitable target for characterizing skin bacteria, our team re-designed the V4 primer pair and tested in silico its ability to improve the coverage of underrepresented propionibacteria. With these new candidate primers, we are able (theoretically) to increase the coverage of Propionibacterium to over 67%–from less than 3%–without losing coverage of the other bacterial groups. Our next step is to evaluate the accuracy of this approach using a mock community as the standard.

There are advantages to using existing V4 primers. They can detect the genera Finegoldia and Peptoniphilus, which are increased in persons with primary immunodeficiencies (Oh et al ., 2013). Zeeuwen et al. have also pointed out that the 27F primer used for the V1-V3 region inefficiently amplifies Gardnerella and Lactobacillus, which have been found to be associated with females (Zeeuwen et al., 2012). In general, V1-V3 classifies fewer populations down to the genus level (Meisel et al., 2016). Because the V1-V3 region is longer than the V4 region, paired-end reads generated with the Illumina MiSeq will not fully overlap. And without full overlap, denoising of reads is not as effective. Using the V3 chemistry (a 600-cycle kit, longer than the 500-cycle kit of the V2 version) will not solve the problem and may even make it worse, because the sequence quality drops after 500 cycles.

In this two-part blog series, we have discussed how to collect enough microbial biomass to run a skin microbiome study, and how to deal with environmental contamination. We have seen that even relatively minor changes in primer sequences may improve the detection of bacteria relevant to skin microbiomes. Feel free to reach out to our team for more information on designing your own skin microbiome study!

About the company

Microbiome Insights provides state-of-the art microbiome analysis and bioinformatics.

Our end-to-end service starts with experimental design and sample collection and extends to data analysis and bioinformatics interpretation.

Microbiome Insights is focused on providing our clients with a deeper understanding of functions and interactions of microbial communities across a range of human, animal, agricultural, and environmental research applications. Our team of experts and testing methods combine to provide fast, dependable, cost-effective results with highly comprehensive, publication quality bioinformatics. To learn more, see here.

How to Design a Skin Microbiome Study, Part I: Sampling

In this post, the first of a 2-part series on skin microbiome research, we will discuss technical issues surrounding sampling of human skin microbes.

Let’s say a researcher sets out to study bacteria on the human skin—the body’s largest organ, which is teeming with microbes from each domain of life, and viruses. The scientific question has been identified and the funding to conduct a pilot study has been secured. Perhaps her group has some experience studying the gut microbiome. For the most part, she has discovered, getting bacteria out of stool is not too difficult; a small amount of material contains enough microbial DNA to sequence the most prominent members of the microbiome. But unlike the intestine, the skin does not support a high-biomass microbiome. If microbes on the skin are present in low abundance, how does our researcher decide on reasonable sampling and sequencing strategies that together capture a representative picture of bacterial diversity?

Credit: stevanovicigor. Thinkstock image used with permission.

Before collecting samples, the researcher must consider key advantages and limitations of available sampling protocols. Commonly used methods involve variations of swabbing – the repeated rubbing of a defined area of the skin with a sterile, pre-moistened swab. When the objective is to obtain sufficient microbial DNA from skin sites with variable and/or low microbial biomass, swabbing can be complemented with scalpel scrapping (Oh et al., 2014). If access to deeper layers, including the dermis, is required, punch biopsies are a viable alternative, but require specialized expertise and are more invasive, reducing the number of sites that can be sampled from the same subject.

Because each method samples a slightly different environment, we would expect different microbial profiles to arise from variations in sampling method. This prediction has not been thoroughly tested (but see Chng et al., 2016). As part of ongoing efforts to improve sampling methods, the Microbiome Insights team is currently evaluating whether D-Squame and Sebutape tape stripping, used for peeling off epidermal layers and sebum, respectively, provide a reliable means of sampling skin microbes. Compared with swabbing, tape stripping recovers ~2- to 3-fold less bacterial DNA. We have yet to evaluate, via amplicon sequencing, whether lower bacterial yield results in different microbial profiles. We are also exploring if coating pre-wetted swabs with aluminum oxide particles maximizes bacterial DNA recovery. The results of this experiment will be made available in a forthcoming technical note.

Handling samples with low microbial biomass is challenging. Even if the sampling method affects how much microbial biomass is collected, the amount of DNA recovered from skin is always low. (Of course, the DNA extraction method affects DNA yield and microbial composition from study to study. But because most studies are comparative in nature, methodological consistency is vitally important.) As most of the DNA is human, obtaining enough genetic material for microbial profiling can be difficult. Microbial load can be increased by instructing participants not to wash with soap or bathe at least 24 hrs prior to sampling, although the effect of cleansing is probably minor compared with the combined influence of sampling and extraction.

Another challenge of low microbial biomass samples is dealing with environmental contamination. Contamination can be introduced during sample collection, DNA extraction, and sequencing library preparation. For instance, bacterial DNA is often found in DNA extraction kits and in other reagents used for preparing samples. And while it is tempting to create lists of “usual suspect” contaminants, this may be futile when studying skin microbes or other human-associated bacteria because, for example, Staphylococcus, a common skin inhabitant, and Escherichia have been identified as potential kit reagent contaminants (Salter et al., 2014).

Processing negative controls alongside low-biomass specimens is critical, because the proportion of microbial DNA attributable to contamination is higher in low-biomass samples compared with high-biomass samples. Usually, we include at least four replicates for each of two types of negative controls on each 16S sequencing run: (1) DNA extraction controls, to assess if kit reagents carry a detectable signal, and (2) template-free PCR blanks, to pinpoint contamination that may arise during downstream processing. For skin microbiome analysis, sterile swabs opened at the site of sample collection are co-processed with the swabbed samples. In general, the number of sequencing reads in our negative controls is about 3- to 4-fold lower than the average in samples derived from skin sites. This is what we would expect for samples containing little to no DNA.

Stay tuned for the second post in this series: amplicon sequencing in skin microbiome studies.

About the company

Microbiome Insights provides state-of-the art microbiome analysis and bioinformatics.

Our end-to-end service starts with experimental design and sample collection and extends to data analysis and bioinformatics interpretation.

Microbiome Insights is focused on providing our clients with a deeper understanding of functions and interactions of microbial communities across a range of human, animal, agricultural, and environmental research applications. Our team of experts and testing methods combine to provide fast, dependable, cost-effective results with highly comprehensive, publication quality bioinformatics. To learn more, see here.

Microbiome Insights’ Year in Review: 2017 by the numbers

This year, 2017, will go down in Microbiome Insights history as the year the company hit its stride and established itself as a leader in the microbiome testing field. The challenging work our team has been doing since we were founded in 2015 clearly paid off—as evidenced by our expanding client list, our strong financial position, and our team’s awards and recognitions.

In the past twelve months we have had to more than double the size of our Vancouver-based team, hiring some of the best technicians, scientists, and bioinformaticians to meet the needs of our clients.

 

Here are some highlights of our company’s 2017, by the numbers:

 

Place out of 150+ companies in the 17th annual BCIC-New Ventures Competition, the largest and longest-running competition for tech companies in BC. The judges, who award $300,000 in cash and prizes to early-stage start-up companies, honoured Microbiome Insights with second place overall and with the Center for Drug Research and Development (CDRD) Life Sciences prize.

 

 

Number of conferences Microbiome Insights attended as exhibitors or sponsors. Our team enjoyed meeting new and existing clients at important academic and industry conferences in all corners of North America—from ASM Microbe 2017 in New Orleans to the Microbiome R&D and Business Collaboration Forum in San Diego. We also trailblazed as one of the first microbiome-centered companies to attend two genomics events: ASHG 2017 in Orlando and the 3rd annual Understand your Genome conference in Boston.

 

 

British Columbia (BC) life sciences companies (including Microbiome Insights) on the ‘Ready to Rocket’ 2017 Life Science Emerging Rocket Lista business recognition program that profiles technology companies in BC with the greatest potential for revenue growth. These “Emerging Rocket” companies are recognized as having a clear business model and go-to-market strategy that will prove attractive to investors.

 

 

Number of microbiome studies our team supported this year—across a broad range of human, animal, agricultural, and environmental applications. We are very proud that many of these were repeat clients.

 

 

Number of dollars invested in our company by Genome BC, following successful equity financing. In combination with funds from the company’s recent round of equity funding, this will help build out our new CLIA-certified lab facility, grow our team and capabilities, and develop and launch new services and tests in 2018.

 

Here are some of the technical insights from our team this year:

  • The staying power of amplicon-based analyses: For studies whose primary objective is to get a comprehensive picture of the bacteria in an environment, or to build models of disease classification (for many diseases) data derived from 16S genes is as good as it gets
  • The power of multi-omics integration: Although it still poses technical challenges, integration of multiomics datasets can reveal things far beyond what each technology can show separately

And finally, here are our team’s picks for the hot areas to watch in microbiome science in the coming year:

  • The contribution of microbes to “inflammaging”—the progressive increase in pro-inflammatory status that occurs with age
  • Progress toward microbiome therapies that modulate the brain through the gut-brain axis
  • Larger and longer studies on the skin microbiome, looking at lifestyle factors and making more definitive associations between skin microbes (composition and function) and healthy skin
  • Tracking the presence of viruses in the gut microbiome and gaining insights about health implications

A very happy 2018, from our team to you!

 

 

How close are microbiome-modulating therapies that target the brain? A quick overview of the evidence

Debate exists about how soon knowledge about the gut-brain axis will bear fruit. Yet the microbiome-gut-brain axis is a hot topic of scientific investigation and several companies around the globe are actively pursuing gut microbiome therapies that focus on brain-related conditions.

Here’s a quick overview from our lab scientists on various areas of brain health and the evidence linking each one to the gut microbiota.

General early life neurodevelopment

Dozens of human studies and mechanistic animal studies support the relevance of gut microbiota to normal behaviour and neurodevelopment; however, these studies are not always specific to neurological development, and the observed effects could be confounded by many other factors that affect the early life microbiome.

Autism spectrum disorders

Although there are known genetic contributors to autism spectrum disorders, both human and animal studies show a connection between gut microbiota and both gastrointestinal symptoms and social deficits in these individuals.

Anorexia nervosa

A moderate level of evidence links anorexia with gut microbiota; no mechanistic studies have been completed to date.

Attention deficit hyperactivity disorder (ADHD)

A low level of evidence implicates gut microbiota in ADHD; this disorder may also be linked to diet, but much more research needs to be undertaken.

Multiple sclerosis

A growing number of human studies as well as mechanistic animal studies have found the gut microbiota has immunomodulatory effects that may affect multiple sclerosis (MS) disease progression. Transfer of the microbiota from a human with MS to a mouse increases MS-like symptoms.

Post-traumatic stress disorder (PTSD)

Moderate evidence and one human study connects the gut microbiota with PTSD; further research may explore the mechanistic role of chronic inflammation as well as cortisol and dopamine regulation.

Depression

A high level of evidence links gut microbiota with depressive symptoms; probiotics may improve depression in both humans and animals.

Anxiety

While the studies on anxiety overlap with those on depression, some reports in both animals and humans show potential of microbiota modulation — for example, through probiotics — for improving symptoms of anxiety.

Fatigue

Extreme fatigue may also be linked with the gut microbiota, although diet appears to be a major confounding factor and more research is required.

Parkinson’s disease

Many studies in humans link Parkinson’s disease (PD) with the gut, but chronic constipation in those with PD is a possible confounding factor. Mechanistic evidence to back these findings is just beginning to emerge.

Alzheimer’s disease

Emerging evidence shows the Alzheimer’s-gut connection: in mice, Alzheimer’s-like symptoms are altered by microbiome manipulation.

A round table discussion at the Global Engage Microbiome R&D and Business Collaboration Forum on Thursday, November 2nd, led by CEO Malcolm Kendall, will explore what we know about the gut-brain axis and how soon it could yield breakthrough therapies.

microbiome insights CEO
CEO Malcolm Kendall

About the company

Microbiome Insights provides state-of-the art microbiome analysis and bioinformatics.

Our end-to-end service starts with experimental design and sample collection and extends to data analysis and bioinformatics interpretation.

Microbiome Insights is focused on providing our clients with a deeper understanding of functions and interactions of microbial communities across a range of human, animal, agricultural, and environmental research applications. Our team of experts and testing methods combine to provide fast, dependable, cost-effective results with highly comprehensive, publication quality bioinformatics. To learn more, see here.

From the human genome to the human microbiome: Toward clinical applications

Genetics versus Genomics

In 1991 the Human Genome Project—a collaborative effort to map the whole human genome—was established. A 5-year plan was put in place addressing the initial framework for the efforts including reliable testing methods, validated protocols, and milestones along the way. This marked a different path from previous studies of genetics—that is, the study of genes, or rather the identification of a particular gene that may be instrumental in a phenotypic outcome. Much of the work in this field had previously been exploratory in nature, with a growing body of evidence linking certain genetic variations or single nucleotide polymorphisms (SNPs) to disease states.

In mid-2000 it was announced that the Human Genome Project had published their results of the almost completely sequenced human genome. While the results were interesting, the data were a far cry from being applicable. What it did do was spur further interest in developing better technologies that would allow cheaper and faster sequencing to add onto these initial findings.

In the years spanning 2004 to 2014 a multitude of companies were competing to churn out faster and better technologies such as the Roche 454 and the Illumina sequencing systems. The technologies were proving to be advantageous in many ways; for example, iterations of these technologies were serving to advance the microbiological sciences.

DNA samples are loaded to 96-well plate for PCR analysis

Awareness of the Microbiome

While the whole genome studies were mushrooming during this decade, the study of microbes was still largely based on culture dependent techniques and there was very little information or interest in communities of microbes residing in the body. Basic microbiology was built on the identification of single pathogenic microbes that were instrumental in disease states, while the non-pathogenic microbes were believed to lie dormant. However, certain areas of research focused on how microbes might influence host, or vice versa.

It was becoming widely accepted that microbes in the gut had a part to play in localized gut related diseases such as Crohn’s but it was less understood how the commensal bacteria shifted in abundance, and what caused these ideal growth conditions. This curiosity began to blossom, largely due to the advances in technology brought about by the human genome project, that would allow these growing questions (and concerns) to be addressed affordably and quickly. In 2007 the Human Microbiome Project was born.

“The recent emergence of faster and cost-effective sequencing technologies promises to provide an unprecedented amount of information about these microbial communities, which will bolster the development and refinement of analytical tools and strategies.”

– NIAID Director, Anthony S. Fauci

Microbial Snapshots

Once it was established that the microbiome was of interest, and of importance to the host, researchers developed new methods for studying it by taking advantage of the high throughput sequencing technologies that came to market during the genomics boom. First amplicon sequencing methods and later shotgun metagenome methods were the gold standard in microbiome research. But scientists began to acknowledge several factors as information about different ecosystems was being compared; first, that microbiomes were specific to their locations and diverse in nature, making them quite different from one body site to another. This was a paradigm shift as many had not considered this level of diversity in commensal and pathogenic bacteria, but also as compared to receiving the same genetic information from every host cell regardless of its location in the body. Secondly, the microbiomes are ever shifting and, upon collection, must be stabilized in such a way that the ‘snapshot’ is maintained at time zero. This means that factors such as temperature and moisture could quickly change a microbial profile if the sample is not treated with care. This opened the doors to a wide variety of collection devices and stabilization buffers with specific media to help maintain these profiles while being interoperable to laboratory procedures.

Clinical Applications

We already see clinical and diagnostic applications for microbiome findings. Although we are still working towards scientifically validating these applications we seem to be on a similar trajectory as we saw with genomics research in terms of diagnostic applications, publications, and consumer-friendly offerings. Interestingly, a singular ‘omics’ (i.e. proteomics, metabolomics, genomics, microbiomics) is informative on its own, but combining multiple features to define functionality of systems in the body will prove to be more fruitful in the long run. Understanding the complex nature of these systems and how they interact will enable us to see how changes or shifts in one system can have effects in other systems. This multi-omics approach is the basis for personalized medicine and furthermore can apply in other domains such as plants, animals, and environmental ecosystems.

At Microbiome Insights we are working with researchers to elucidate synergistic effect of multiple ‘omics’ at work. With this approach we are focused on the skin microbiome, the gut-brain axis, pharmacology, and other areas of science that bring together the genome and microbiome for a better understanding of human health.

About the company

Microbiome Insights provides state-of-the art microbiome analysis and bioinformatics.

Our end-to-end service starts with experimental design and sample collection and extends to data analysis and bioinformatics interpretation.

Microbiome Insights is focused on providing our clients with a deeper understanding of functions and interactions of microbial communities across a range of human, animal, agricultural, and environmental research applications. Our team of experts and testing methods combine to provide fast, dependable, cost-effective results with highly comprehensive, publication quality bioinformatics. To learn more, see here.