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Microbiome Insights scientific advisory board member Curtis Huttenhower contributes to research identifying thousands of new human microbiome species

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Thousands of new microbial species making up the human microbiome have been identified from metagenome samples collected around the world. By reconstructing the microbial genomes found in over 9000 metagenome datasets, the microbial genomes of the unnamed species extend the knowledge of the human microbiome and should aid in development of future metagenomics technologies.

The work of identifying these new species was carried out by a team of researchers from Europe, New Zealand and the US, and included Harvard’s Dr. Curtis Huttenhower (a Microbiome Insights scientific advisory board member). The group used a scalable bioinformatics methodology to reconstruct the genomes of unknown bacteria found within the metagenome assemblies. Here the metagenome samples were site-specific human body samples (oral cavity, skin, vagina, and stool) from multiple people living all over the world. The diverse set included individuals of all ages, living varied lifestyles from 32 countries.

A wealth of information is contained in these samples, as they represent the whole-body microbiome of humans from different geographic locations that experience different climates and circumstances. Seven of the datasets also came from non-Westernized environments, further expanding the range of conditions the microbes are sampled from. Using these metagenomes as the starting point the researchers were able to apply their large-scale single-sample metagenomics assembly and identified 4930 species-level clades, 77% of which had no previous whole-genome level information.

Their method of assembly was optimized to maximize the quality of the microbial genomes being found in the metagenome sample, rather than the quantity. Despite this strict method, 154 723 new microbial genomes were identified, which more than doubles the current publicly available set of roughly 150 000 microbial genomes. With this investigation having doubled the catalogue of known microbial genomes, future studies attempting identify the contents of a metagenomics sample now have a more comprehensive reference set from which they can map out their samples. The metagenomics assembly strategies used in this work can also be applied to non-human associated metagenomes and will be applicable for new sequencing technologies such as synthetic or single molecule long read sequencing.

A large fraction of the previously unidentified species were seen in the non-Westernized samples; however, examples of the new species were prevalent throughout all samples. Roughly 2.5 million genes were also found within the known species-level clades, many of which were associated with conditions including infant development and Westernization. Several taxa of bacteria were found to be prevalent in this analysis despite not being observed in previous well-profiled populations. Still more taxa from underrepresented phyla, such as Saccharibacteria and Elusimicrobia, were found in oral and gut microbiomes.

According to the study authors, “the resulting genome set can thus serve as the basis for future strain-specific comparative genomics to associate variants in the human microbiome with environmental exposures and health outcomes across the globe.”

Pasolli E, Asnicar F, Manara S, et al. Extensive unexplored human microbiome diversity revealed by over 150,000 genomes from metagenomes spanning age, geography, and lifestyle. Cell. 2019; 176: 649-662.

 

 

 

As founding sponsor of Hanson Wade Skin Health & Dermatology conference, Microbiome Insights speaks about skin microbiome study design

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With its globally leading expertise in skin microbiome testing, Microbiome Insights was a proud founding sponsor of the Skin Health & Dermatology Conference, held in San Diego September 10th to 12th, 2018. According to conference organizers Hanson Wade, the aim of the event was “understanding the underlying biology of the skin microbiome for translation into safe, effective, and commercially viable dermatological therapeutics & cosmetic products”. Participants heard about microbiome-focused skin products already on the market and those under development by companies around the world.,

Microbiome Insights CEO Malcolm Kendall spoke at the conference, with a presentation entitled: “Swab to Data – Considerations for Designing Skin Microbiome Studies”. The talk covered expertise developed by the Microbiome Insights team by working with leading scientists and industry partners in cosmetics and dermatology, and explained how the company has developed new 16S V4 region primers for improved skin microbiome analyses.

Also on the speakers’ list was Greg Hillebrand, Senior Principal Scientist at Amway, who spoke about a study carried out in partnership with Microbiome Insights: “Temporal Changes in the Facial Skin Microbiome: A One-Year Longitudinal Study in Normal Healthy Men and Women”.

To find out more about the conference, see here.

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

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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?

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., citing previous work, 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!

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

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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?

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.

At Understand Your Genome event, Microbiome Insights participates in latest discussions on genome sequencing for disease prediction and prevention

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Microbiome Insights Inc. was an exhibitor and sponsor at the third annual Boston Understand Your Genome conference, held on Nov. 14, 2017. The all-day event centered on the progress and promise of genomic medicine and the issues regarding sequencing the genomes of healthy individuals for disease prediction and prevention.

The conference topics included sequencing and informatics in clinical care, precision health, and understanding the basics of genetics and genomics. Among the event’s speakers were Dr. Hannah Valantine, Chief Officer for Scientific Workforce Diversity at the National Institutes of Health, who discussed racial disparities in organ transplant outcomes and diversity issues in genomics; and Dr. Calum MacRae, Chief of Cardiology at Brigham and Women’s Hospital in Boston, who spoke about the need for a robust global phenotype effort to replace the outdated diagnostic guidelines used today.

All conference participants were also offered the opportunity to have their genome sequenced and analyzed for genes and variants associated with adult-onset conditions, carrier status, and drug response.

Microbiome Research: Don’t Forget The Fungi

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Microbial Interactions In Living Systems

The emerging field of the microbiome is in pursuit of understanding the microbe-microbe and microbe-host interactions that occur in virtually all living systems. This includes interactions critical to plant and animal health. The fundamental question is: What role(s) do the microbes and their functions play in broader systems?

Advancements in technology, such as next generation sequencing, have allowed us to overcome the barriers of culture-dependent methods of identification and classification, providing the ability to sequence and identify a more complete community of microbes in any given sample with a high degree of sensitivity and reproducibility.

It seems, however, that the term ‘microbiome’ tends to implicitly refer to commensal and pathogenic bacteria, with very little attention paid to the role of eukaryotic organisms. As such, the field is heavily utilizing 16S amplicon sequencing to increase our understanding of these bacteria. But what about other amplicons such as 18S or ITS (internal transcribed spacer) that shed light on eukaryotic or fungal communities?

Penicillium, ascomycetous fungi of major importance

The Mycobiome

In recent years, there have been more published data elucidating the presence and contribution of fungi in certain disease states. These fungi have been shown to interact with bacterial communities in either a synergistic or competitive manner. In either relationship, these fungi may be a critical component of the progression of such diseases—including hepatitis B, cystic fibrosis, and even inflammatory bowel disease. As a result, researchers have coined the term ‘mycobiome’ to refer to the communities of fungi that may play an interesting role in the system.

The human body is a unique ecosystem, and we have long known mucosal sites are abundant with fungal flora.  What we are beginning to identify is how these communities interact with other sites across the body, where bacterial communities reside. Besides the specific diseases associated with fungi, we are seeing evidence that overall gut health is maintained by a degree of fungal-bacterial interaction. Mechanistically, the mycobiome appears to play a role in inflammation and metabolism by modulating the bacterial microbiome.

Environmental microbiomes are also of growing interest in the microbiome field, with a particular focus on ocean microbiomes, air pollutants, and soil microbiomes. In soil microbiomes, where fungi are prevalent, researchers are interested in certain subcategories; studies focus on the soil microbiome, the plant/rhizosphere microbiome, or the point at which these two microbiomes interact, which is called the mycorrhizosphere. The fungal component of these microbial communities plays a critical role in the nutrition and growth of plants as well as in the exclusion of plant diseases. Mycorrhizal fungi have even been shown to mediate signaling between plants.

Amplicon Sequencing: 16S, 18S, & ITS

Unlike other next generation sequencing labs that have exclusively focused on the approaches of molecular biology and genomics, Microbiome Insights also pulls from expertise in microbial ecology and infectious disease to provide a more complete picture of the ecosystem. We have implemented standardized protocols for 16S (Prokaryotic) sequencing and have developed robust workflows for both 18S (Eukaryotic) and ITS2 (fungal) sequencing using the Illumina Miseq.  We apply these approaches as necessary to address specific scientific questions, and can build on this data using shotgun metagenomic sequencing. Altogether our expertise allows us to provide our customers with a full suite of tests—without forgetting the fungi.