Drawing meaningful conclusions from microbiome experiments requires certainty in the experimental design and subsequent analysis; appropriate statistical descriptions of the microbiome are still an emerging area of inquiry. Dr. Tonya Ward, Diversigen’s Director of Data Science & Bioinformatics has worked throughout her career to identify methods to better characterize the microbiome. She has previously profiled the developing mycobiome from birth onwards1 and has linked antibiotic treatment to microbiome dysbiosis and disease2.
Prior to joining Diversigen (formerly CoreBiome), Dr. Ward characterized the human milk metagenome and function of human milk immune proteins at the University of Ottawa. Afterwards, she joined the lab of Dr. Dan Knights at the University of Minnesota-Twin Cities – where she researched new analysis tools for microbiome research and completed clinical and experimental microbiome studies. Dr. Ward helped shift the understanding of human enterotypes3 and improved microbiome classification systems. Her work has even identified how US immigration can westernize the microbiome.4
Next in our “Behind the Data” series profiling Diversigen microbiome expert, Dr. Ward we talk about the microbiome and how Diversigen is pioneering the future of bioinformatic applications. Here is a re-cap of our discussion:
The human microbiome forms at birth as microbes colonize newborn babies; can you tell us more about the formation of the human microbiome?
Dr. Ward: Many factors influence the formation of the human microbiome; things like birth mode, type of feeding, the mother’s microbiome, and even the developmental environment will all play a role in the formation of a babies’ microbiome. Although the human microbiome is a dynamic system that can change overtime, the succession is actually quite well ordered. It is even possible to predict the age of an infant based on the composition of their microbiome.
Ordered microbiome development is also not exclusive to mammals; other non-mammals also show a predictable formation of their microbiomes. This means while the formation of a microbiome is dynamic, common factors are widespread across life that create similar patterns throughout microbiome development.
Children are commonly prescribed antibiotics which can cause pediatric dysbiosis. How do these medications impact early human health and disease?
Dr. Ward: Antibiotics are like a forest fire to microbiome ecosystems – a single dose of antibiotics can cause a huge disruption removing a large percentage of microbes. After a single dose, the human microbiome can generally recover once the body has cleared the antibiotic. However, continuous exposure can lead to problems – especially in infants. Continued suppression of the microbiome disrupts proper microbes from establishing themselves and in infants the ordered succession of microbiome development makes it critical that the right microbes colonize within the proper time frame*.
Disruption of infant microbiomes has been observed through longitudinal studies; by collecting information and studies from children over several years, researchers are able to create a clear picture of infant microbiome development and health trends. Correlations have been made between phenotypic outcomes like allergies and the establishment of certain taxa in their developmental time frame.
While exploring the mycobiome in infants, you’ve observed the dynamic fungal community in early life, and have examined the fungal taxa inhabiting infants. What is the mycobiome? How does the mycobiome impact human health?
Dr. Ward: The mycobiome is all the fungi associated with the host, and a component of the total microbiome. Fungi can be observed similar to bacteria by using amplicon sequencing, where bacteria have a universal 16S gene, and fungi have a universal ITS2 gene. The mycobiome can also play a role in health. Like bacteria, specific fungal taxa can be linked to phenotypic outcomes later in life;, for example, the genus Pichia is associated with allergic outcomes. Fungi also influence the microbiome community; bacteria, fungi, and viruses cohabitate in the microbiome balancing each other and their environment. The disruption of any one species can cause a spiral leading to dysbiosis as taxa can be dependent on each other.
Observing the mycobiome in early life across various sites, you’ve studied the microbiome from various sample types including oral, anal, and skin samples. How does sample collection across different sites impact microbiome research?
Dr. Ward: Different sites like oral, anal, and skin give an encompassing picture of different areas in the body where the microbiome plays a role. However, the approach and techniques used can be dependent on the specific sites you are studying. For example, it can be difficult to obtain enough bacterial or fungal DNA from low biomass samples like skin swabs and without significant amounts of microbiome DNA, performing shotgun sequencing would require extreme depth to overcome contamination in the samples from host DNA.
Additionally, if one were to sample across multiple sites, it becomes critical to pair sequencing technologies and analysis that are viable across all samples and conditions. Each method can introduce bias and errors and controlling for these throughout sampling and sequencing is vital to finding trends or significant signals in the data. Failing to connect methods and technologies would result in the information between samples becoming very difficult to compare.
Diversigen has researched the human gut microbiome, the skin microbiome, the animal microbiome, the mycobiome, and, now, the virome – Which projects stand out to you that helped define the field?
Dr. Ward: Diversigen recently collaborated with Dr. Jasmohan Bajaj at the Virginia Commonwealth University on his investigation of bacteria, phages, and their role in liver disease severity. In the study it was observed that phage bacteria interactions are altered by cirrhosis treatment, indicating people receiving antibiotics were not only shifting the bacterial community, but also the viral community. This study stood out to me because the phages existing in the microbiome were affected by the changing abundance of bacteria from an external exposure. The virome, while less understood, has potential for new discoveries that could help explain the microbiome’s role in human health.
In one of your papers you helped describe how captivity humanized the primate microbiome. How does this impact microbiome research on humans and primates?
Dr. Ward: The discovery that the primate microbiome is humanized by captivity goes beyond human and primate research and impacts all hosts in microbiome research. Consider any host under study – their microbiome is dynamic and many influences from environment, age, diet and exposures all impact the microbes that exist within their specific microbiome. We can translate that to experimental research in the sense that the specific microbiome of a model organism, like mice, may or may not be considered in study design but will play a huge role in the outcomes. For example, testing a treatment or antibiotic on a mouse model may show taxa shifting, however, this may not hold true if the mice were wild, or in a different host with a different endogenous microbiome.
It is crucial to know all the upstream confounding variables that can impact the community under research because whenever possible, we try to account for variables in upstream study design and subsequent analysis. Maximizing metadata on samples by including information like age, exposures, diet, and location allows analysis to account for other bias and errors that may impact the ability to identify significant relationships between taxa and outcomes.
You helped show that enterotypes are fluid and continuous, which sparked discussions on rethinking enterotypes. What are enterotypes? And how might their use have an impact on microbiome research?
Dr. Ward: Enterotypes are defined clusters microbiome profiles based on similarity. Blood types, for example, have specific types like A, B, O, which were created to define the different categories and traits of blood between people. Following this model, the microbiome community created enterotypes as categories for microbiomes. However, unlike blood types, enterotypes change over time as the microbiome changes. The potential daily shift of thousands of microbial community members makes attempting to create discrete categories quite difficult. Personally, I believe the microbiome is more of a spectrum, a moving target that can change from day to day, and enterotypes can be misleading as an individual microbiome could potentially shift from type 1 to type 3 over time. Although, the gold standard for the microbiome is always evolving as we continue to learn more and reconsider what is the “right” way to define the microbiome. That’s why developing methods to understand the microbiome is important as there is always a chance for new discoveries.
When helping to demonstrate the impact of antibiotics or chemotherapy, you used shallow shotgun [BoosterShot] sequencing to identify the loss of key ‘health-promoting’ bacterial species and altered functional profiles in mice. Can you tell us why this sequencing method was chosen?
Dr. Ward: Shotgun sequencing allows researchers to get better specificity and observe what members are within the microbiome – allowing us to see the species and strain level of bacteria. This kind of resolution can play a big role in finding biomarkers. Being able to identify these key species in the microbiome is critical to understanding the impact of treatments like antibiotics and chemotherapy. Patients lacking protective members of their microbiomes can be at risk, and if we could sample individuals prior to treatment there is potential to reestablish key community members before treatment for a better outcome.
Sometimes a signal can come from a specific strain of bacteria which might otherwise go unnoticed at the genus level that is observed using amplicon sequencing. When thinking about study design, to get more power you usually need more samples to sequence, which of course, means a higher budget. However, by using shallow shotgun approaches you can get high specificity with lower costs compared to deep shotgun methods. Microbiome studies can get more power from a greater amount of data, and generating this data can come from increased sample size or a higher depth of sequencing.
Microbiome experiments often begin with sample collection. Can you tell us how Diversigen helps researchers plan their projects and decide on which samples to study, and how to sequence them?
Dr. Ward: The team of bioinformaticians at Diversigen is quite large and each person has an inherit knowledge of the microbiome from their own research in the field. Our bioinformaticians are trained in both the dry and wet lab and know how to design a microbiome study to draw the most powerful conclusions. We are more than happy to work with our customers to optimize their study designs, planning upstream processes like sample collection and preservation with downstream sequencing and analysis to get the best out of their experiment. There are potential pitfalls when planning a microbiome experiment and sometimes key choices upstream can play a big impact downstream. For example, collecting samples in different preservation buffers can introduce a large bias into the data. By partnering with microbiome experts, we ensure that considerations are made at the start of a project to maximize study results and analysis.
You helped contribute to the research study titled, “US Immigration Westernizes the Human Gut Microbiome”, what did you learn about how immigration affects the microbiome?
Dr. Ward: The microbiome is very dynamic and similar to how the microbiome develops from infancy onwards, there are clear links between diet and environment that influence how your microbiome is shaped. When a person moves to a drastically different environment, like from south-east Asia to the US, their microbiome begins acquiring new microbes and sheds others. When an individual immigrates to the US, their diet is likely to change drastically and combined with a shift to an urban environment, there can be a huge impact on the types of bacteria found in the GI tract. In infancy, the host’s immune system does not overreact to the microbiome changes as they develop together, however, when dramatic shifts happen later in life, there is potential for the immune system to react. Understanding these host-microbe interactions and how they affect outcomes is still ongoing, but there is growing evidence showing the role of the microbiome in diseases like obesity. Expanding research to the individual level will be critical to understanding these connections and one day finding personalized responses and treatments.
How is Diversigen continuing to support research into human health?
Dr. Ward: Diversigen collaborates with some of the best researchers in the microbiome field today. We work closely with our customers to design the best studies and return the highest quality data and annotations. Our labs are high-throughput, allowing us to expand research to a huge scale. We hope that by resolving individual microbiomes with greater detail, Diversigen will contribute to discoveries into the human microbiome and health interactions.
Dr. Tonya Ward has helped establish crucial bioinformatic platforms for microbiome analysis and has published research observing the development of the human microbiome from birth to adulthood. Her research has linked age related microbiome developments to health and disease states and she has gone on to apply her own discoveries into creating Diversigen’s unparalleled bioinformatic analysis platforms.
Our microbiomes can describe us better than ourselves sometimes. Understanding microbiome development helps researchers link changes between the microbiome and host health. Diversigen experts have studied the microbiome at every stage of development and are ready to help accelerate your microbiome discoveries.
Ready to talk to Diversigen microbiome experts like Dr. Ward? Our bioinformaticians are ready to help you plan your project, visit us at www.diversigen.com or send questions to info@diversigen.com
- https://msystems.asm.org/content/msys/3/3/e00140-17.full.pdf
- https://www.sciencedirect.com/science/article/pii/S193131281500164X
- https://www.sciencedirect.com/science/article/pii/S1931312814003461
- https://www.sciencedirect.com/science/article/pii/S0092867418313825
