It's no secret that the microbiome is getting traction every day with new research and discoveries. Being part of the discussion and performing studies in this important area of science shouldn't be a challenge. That's why our team created our new comprehensive Microbiome Study Guide.
Our goal is to support anyone interested in studying the microbiome and facilitate the process of designing and executing a study.
What is a Microbiome Study?
A microbiome study focuses on answering a research question or testing a hypothesis by analyzing the microbial composition (or the microbiome) on different samples. Usually, investigators have primary and secondary objectives that could lead to follow-up hypotheses or may require additional investigation to gain a more detailed understanding of the research topic.
The usual steps taken on conducting a microbiome study are:
- Storage and shipment
- DNA extraction
- Profiling, including DNA sequencing and/or metabolomics
- Bioinformatics, data visualization and reporting
Why is Microbiome Research important?
A microbiome study or research can have multiple applications like testing how a diet changes the gut microbiome, validating the efficacy of a probiotic, supporting a health claim or testing a product for its impact in different microbiomes (skin, oral, gut, etc).
Microbiome Sample Types & Collection Methods
There is a broad range of samples that can be collected for microbiome studies. Some sample types that are commonly studied in microbiome research include:
- Fecal samples
- Skin samples
- Oral samples
- Urine samples
- Vaginal samples
- Soil samples
- Water samples
There are specific ways to collect, store and ship each sample type. The primary considerations when sampling are using the correct sampling device, preserving samples under adequate storage conditions, being mindful of sample quantity and shipping under appropriate guidelines.
This step can be performed either in-house or by outsourcing it to another lab or CRO organization. It is important to note that DNA extraction is very susceptible to bias, and as such, it influences the analysis results.
Microbiome Sequencing Options
There is a range of sequencing options available, each method having its pros and cons. Typically, these are the kinds of analysis you can expect from a lab specialized in microbiome-based studies:
rRNA Gene Sequencing of 16S, 18S, ITS2 and archaeal V4-V5
Amplicon sequencing, or rRNA gene sequencing, is performed to determine the relative abundance of taxa in a bacterial community, and to compare between groups of interest. This level of analysis can help to address changes in the overall microbial profile over time, or between treatment groups. Depending on the sample type, the following amplicons are commonly used:
- Prokaryotic communities (16S V4, V1-V3)
- Eukaryotic communities (18S)
- Fungal communities (ITS2)
- Archaeal communities (16S V4-V5)
Total Bacterial Quantification via qPCR (quantitative PCR) for total bacterial content
Quantitative PCR (qPCR), also known as Real-Time PCR, is a method that measures the number of copies of a DNA region defined by a particular PCR primer(s). Using this method, we specifically amplify the 16S amplicon and quantify the total bacterial content in each sample to determine total bacterial load.
Shotgun Metagenome Sequencing
Shotgun metagenome sequencing is taxonomic profiling (diversity and abundance), and functional analysis of a microbiome. This technique allows for parallel sequencing of DNA from all organisms within the community, with high coverage for species-level detection. The data generation allows for more advanced reporting and genome assemblies.
Shallow Shotgun Metagenome Sequencing (SSMS)
Shallow Shotgun Metagenome Sequencing Involves sequencing samples at a shallower depth than is applied in full shotgun metagenome sequencing. By combining many more samples into a single sequencing run and using a modified protocol that uses a lower volume of reagents for sequencing library preparation, SSMS is an economical way to provide compositional and functional sequencing data similar to deep shotgun metagenome sequencing.
Short Chain Fatty Acid Analysis
Short chain fatty acids (SCFA) are the products of fermentation of insoluble fibre from diet (eg. cellulose, resistant starch) by the bacteria in the gut. These fatty acids have been shown to play an important role in regulating metabolism in the gut and are closely associated with gastrointestinal diseases such as irritable bowel syndrome and other conditions such as obesity. By quantifying SCFA in stool, one can monitor gut health and inflammation.
Bioinformatics, data visualization and reporting
Once your sequencing step has been completed, the results are shared as raw sequencing files in a FASTQ format. FASTQ format is the standard for storing the output of high-throughput sequencing instruments such as the Illumina sequencers. From here, there are several subsequent steps required to process the data before you are ready for data analysis and report generation. There are multiple data processing methods, and it is important to have the support of a bioinformatics team.
What is bioinformatics?
Bioinformatics is an interdisciplinary field of science that combines biology, computer science, information engineering, mathematics and statistics to analyze and interpret biological and clinical data.
Data can help us tell the story. We need to keep the audience in mind. Context is important, and so are the design and colours used.
- Amplicon studies usually contain standard bioinformatic reporting including plots showing taxonomic composition, alpha diversity, beta diversity (ordination), differential abundance testing, and multivariate analysis (permutational analysis of variance). This is accompanied by raw bioinformatics outputs (OTU table, taxonomic classification of each OTU), differential abundance results, and relative abundances of taxa at various taxonomic levels.
- Metagenome sequencing reports will contain high-resolution taxonomic and functional profiles analyzed observing the same principles that guide the analysis of amplicons.
- Advanced bioinformatic reporting may include:
- microbial epidemiology
- multiple regression
- partitioning sources of variation
- multi-omics integration analysis
- clustering algorithms
- machine-learning-based modelling
- biomarker discovery
Get started in microbiome research
To get started, determine which type of microbiome you want to analyze, and develop a research strategy to begin your research today. Use our guide as a reference and reach out to us for any support you need on the way.