1. Microbiome studies for microbial disease pathogenesis research
Jennifer Myskiw
Msc Student in Dr. Stephanie Booths Lab
NML

2. What is a microbiome?
Group of microorganisms or microbes that inhabit a particular environment
May consist of bacteria, viruses, and/or fungi
Can be symbiotic or pathogenic
Play a significant role in human disease and health
Gut Health at every age, Abbot Global, 2017 <

3. Human Microbiomes Extremely abundant and diverse
Approximately trillion microbial cells living within the human body
The human gut houses the most bacteria in the body and varies from person to person
The human gut plays a significant role to human health and has been linked to many diseases
Unlocking the secrets of the microbiome, The New York Times, 2017 <

4. Microbiome Studies
Microbiome papers published, NCBI, 2017 <
Due to advances in technology and appreciation of how human microbiomes play a role to human health, the amount of microbiome studies have drastically increased

5. Microbiome Study Challenges
Reproducibility is key
Meticulous documentation of how data was collected, processed, and analyzed is necessary
Metadata is an important factor when planning and executing microbiome studies
Confounding factors can skew data leading to incorrect assumptions
Experimental design considerations for microbiome experiment. Retrieved from “Best practices for analysing microbiomes” by R.Knight et al. 2018, Nature, 16, 414.

6. Typical Experimental Workflow
Typical experimental workflow with experimental variables. Retrieved from “Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design” by D. Laukens et al. 2016, FEMS Microbiology Reviews, 40, 119.

7. Experimental Design – Human Studies
Cross-sectional Studies
Observational study at an individual time point
Useful when wanting to find differences between human populations
Longitudinal Studies
Observational study that follows an individual over a span of time
Possible to collect baseline sample
Reduce variables found in cross-sectional studies
Interventional Studies
Experimental study where there is a test group and a compare group (baseline sample)
Allows direct observation between microbiome effect on or from treatment/environment

8. Experimental Design – Animal Studies
More easily controlled and manipulated compared to human studies
Rodents are preferred based on their physiological similarities to humans and them being well defined
Germ Free mice (GF)
Born and raised without exposure to microorganisms
Allow a direct link between specific bacterium and effect
Humanized mice
Microbiome composition very similar to donor human
Limitations include strong species-associated microbiota within the gut
Retrieved from <

9. Experimental Design – Animal Studies
Caution must be used when using rodents for microbiome studies
Research environment (cages) and breeding are confounding facts
Caging of mice, diets of the mice and mothers, and stress factors must all be taken into consideration and documented
Retrieved from “Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design” by D. Laukens et al. 2016, FEMS Microbiology Reviews, 40, 125.

10. Sample Collection and DNA extraction
Human and mice samples often include feces collection or extraction of intestinal tissue
DNA extraction methods may lead to biases, it is important that the same reagent kits be used for all samples within a study
Blank controls should be used when using amplification kits to rule out any potential contamination
DNA Precipitate, 2009, Retrieved from <

11. Sequencing
Different sequencing and analysis methods for microbiome research. Retrieved from “Combing metagenomics, metatranscriptomics and viromics to explore novel microbial interactions: towards a systems level understanding of human microbiome” by S. Bikel et al. 2015, Computational and Structural Biotechnology Journal, 13,

12. Sequencing – Marker Gene analysis
Sequencing with the use of primers that are specific to a gene region
16sRNA is used for bacteria and archaea and internal transcribed spacer (ITS) is used for fungi
Well tested, relatively cheap, and fast
Errors should be removed from sequencing through clustering OTUs or through oligotyping
Taxonomic names may then be designated to microbial communities through databases

13. Sequencing – Metagenomic and Metatranscriptomic analysis
Metagenomic sequencing
DNA sequencing of all microbial genomes found in a sample
Metatranscriptomic sequencing
RNA sequencing in order to understand gene expression and functional activity
Both of these methods allow for in depth understanding of the microbes collected
Collected DNA or RNA sequences should be compared to reference data bases in order to allocate taxonomy
Data may also be organized into short reads forming contigs, by doing so it is possible to explore data beyond taxonomic assingment

14. Case Study
Davenport et al. used a GWAS to investigate genetic contribution to microbial composition within the gut of humans
Worked with Hutterite communities in North America
Looked for differences in gut microbiome composition in regards to age, sex, and genetic affects
184 stool samples collected over summer and winter
Microbial composition was compared to genotype information from each participant
Retrieved from <
Found 8 bacterial taxa whose presence in gut was related to SNPs in the host genome
One of these bacterial taxa was linked to obesity, suggesting some individuals may be predisposed to diseases such as obesity due to genetic factors

15. Significance
Microbial communities within the gut have many functions such as synthesis of vitamins and amino acids, development of the immune system, and resistance to pathogens
Defects in the host genome affecting microbial composition of the gut or disruption of homeostasis in the gut often lead to complex diseases
By furthering our understanding of the relationship between the human body and the microbes that inhabit it, new treatments, therapies and preventatives to devastating human diseases are possible
Main functions of bacteria in the gut. Retrieved from “Heterogeneity of the gut microbiome in mice: guidelines for optimizing experimental design” by D. Laukens et al. 2016, FEMS Microbiology Reviews, 40, 118.

16. Questions?