Exploring the gut microbiome and its impact on our health is an exciting field of research. Thanks to new microbiome testing technologies, we’re now able to examine the gut in greater detail than ever before. Here, we delve into the intricacies of different sequencing technologies, microbiome testing and the future of the field.
What is Gut Microbiome Testing & Why Is It Important?
Have you ever wondered how your gut health affects your overall well-being? Scientists have found that the gut microbiome plays a crucial role in our well-being, due to the composition of both beneficial and harmful bacteria. This has naturally sparked curiosity about learning the health of our own microbiomes, which is why microbiome testing has become such a hot topic recently!
Tracking your gut health through microbiome testing is essential to understand what you can do to support your gut bacteria. Knowing what your gut is made of paves the way for personalized treatment strategies and targeted interventions. But how exactly does microbiome testing work? The secret lies in decoding the microbiome genome through a process called sequencing.
By definition, the microbiome refers to all of the microorganisms in the human gut and their genes. By taking a sample and studying it, scientists can learn a lot about the state of your microbiome. This insight can help you make informed choices to improve your gut health and, in turn, your overall well-being.
What is Sequencing?
DNA sequencing is the process of figuring out the exact order of nucleotides in a DNA molecule. This means identifying the sequence of the four bases – adenine (A), thymine (T), cytosine (C), and guanine (G) – that make up DNA.
Since every organism’s DNA is unique, the specific order of these bases can be used to determine the species. This is how scientists use sequencing to profile the gut microbiome.
Over time, sequencing has undergone numerous revisions and improvements, making it more efficient, accurate, and much faster. Researchers are constantly looking for ways to increase throughput, or the ability to study multiple DNA strands simultaneously.
History of Sequencing
Before a way of decoding DNA was created, scientists had to study microbiomes by taking samples, growing cultures, and examining the bacteria directly¹. Although this method gave us a general idea of the kinds of bacteria in our gut, it couldn’t tell us how much of each species was actually present.
Yet, in the 1970s everything changed. This was due to the introduction of two groundbreaking methods – Sanger sequencing and Maxam-Gilbert sequencing – which were collectively referred to as first-generation sequencing.
Both of these methods worked in a similar way. They involved separating the DNA strand into smaller pieces, separating these fragments from one another, and then individually studying the fragments one at a time.7,8
While these techniques were revolutionary, they were also super time-consuming and low-throughput since each DNA fragment had to be analyzed manually one at a time, without any computer help.
Consequently, it took scientists over a decade to decode the human genome using these techniques. Considering that the collective human microbiome genome is about 100 times larger than the human genome⁹, sequencing the gut would have been impossible without the development of a more efficient sequencing method.
The process of classifying the microbiome has transformed in recent years with the development of modern technologies that allow for faster, high-throughput profiling.
These technologies, collectively known as next-generation sequencing (NGS), work by analyzing multiple DNA strands at once rather than one at a time, resulting in a more efficient and cost-effective sequencing method1. But how efficient are these NGS methods exactly?
The Human Microbiome Project (HMP) was an almost decade-long initiative meant to improve the understanding of bacterial ecology by studying the microbiomes of humans from diverse backgrounds and their overall health. With these NGS methods, researchers were able to sequence the microbiomes from 18 human body sites, which allowed them to characterize over 47,700 bacterial specimens².
Currently, there are two major high-throughput sequencing approaches for microbiome profiling: whole genome shotgun sequencing and targeted 16S rRNA amplicon sequencing.
Current Microbiome Tests
| Microbiome testing technique | Objective | How it works |
|---|---|---|
| NGS sequencing (Shotgun sequencing, 16S rRNA sequencing) | Provides a general composition of the microbiome and possible ways to improve bacterial diversity | Identification and profiling of bacteria in a sample by decoding DNA |
| qPCR | Used to detect and measure suspected bacterial infections | Amplification of specific DNA sequences present in the suspected bacteria |
| Transcriptomics, proteomics, and metabolomics | Measuring activity of bacteria (some bacteria might be in large numbers but may be dormant) | Proteins, genetic products, and metabolic products created by bacteria are extracted from sample and then traced back to the bacteria |
Shotgun Sequencing
Shotgun sequencing has been a game-changer in microbiome profiling, especially since it can handle genomes that are small and diverse. This method involves sequencing all the DNA in a sample, offering a comprehensive view of the microbial community, including both identification and functional analysis of the microbiome⁵.
How Shotgun Sequencing Works³:
- Sample collection: a stool sample is collected from which DNA is extracted using appropriate DNA extraction kits.
- DNA fragmentation: DNA strands are split into multiple fragments simultaneously (either mechanically or chemically).
- Sequencing: the fragments are loaded onto a computer platform where millions of fragments are read.
- Data processing: fragment sequences are reassembled to reconstruct the original DNA strand on the computer in order to derive the entire genome sequence.
- Taxonomic profiling: the species of the bacteria present in the sample is identified based on their unique DNA sequence.
The magic of shotgun sequencing lies in its ability to read millions of DNA fragments simultaneously using computer software, making the process high-throughput. While traditional methods required analyzing each fragment one at a time, shotgun sequencing reads them all at once⁴.
However, the random fragmentation of DNA can sometimes introduce inaccuracies, especially with larger samples, potentially requiring extra sequencing cycles for a precise representation of bacterial diversity¹.
Shotgun sequencing is also computationally intensive due to the vast amount of data that needs to be processed. Isolating and reading the genomes of millions of bacteria can be labor-intensive and costly.
16S rRNA Sequencing
16S rRNA sequencing is similar to shotgun sequencing in that it can analyze multiple DNA strands simultaneously, providing a detailed view of a microbial community⁶.
However, unlike shotgun sequencing, which reads the entire genome, 16S rRNA sequencing targets a specific region within the genome: the 16S subunit. This method is therefore more focused and cost-effective¹,⁵.
How 16S rRNA Sequencing Works:
- Sample collection: a stool sample is collected from which DNA is extracted using appropriate DNA extraction kits.
- Amplification: the 16S subunit is isolated and multiple copies of the subunit are created.
- Sequencing: the fragments are loaded onto a computer platform where millions of fragments are read.
- Taxonomic profiling: the sequence of the 16S subunit is used to identify the composition of the microbiome
The 16S subunit is present in the genome of all bacteria, but its sequence is unique to each species¹. This method works by amplifying only the 16S subunit, reading its nucleotide sequence, and matching it to the species of bacteria.
Since all bacterial genomes contain the 16S subunit, a universal primer can be used to isolate it from all bacteria in a sample, resulting in a straightforward and high-throughput sequencing method.
Microbiome tests often use 16S rRNA sequencing for its cost-effectiveness, while still providing ample information about our gut health.
To recap, here are the key differences between shotgun sequencing and 16S rRNA sequencing:
| Shotgun Sequencing | 16S rRNA Sequencing | |
|---|---|---|
| Pros | High throughput Provides a comprehensive overview of entire genome Can identify genes, pathways, and new organisms | High-throughput More efficient and cost-effective due to targeted sequencing of 16S Reliable for microbiome profiling |
| Cons | More expensive Computationally intensive, rendering it inaccessible for general microbiome testing Possible need for extra sequencing cycles | May not narrow microbiome composition down to the species level, only genus |
Quantitative Polymerase Chain Reaction (qPCR)
While shotgun sequencing and 16S rRNA sequencing aim to get a general sense of the kinds of microbes living in our gut, qPCR is used to test whether a patient has an excess of a particular kind of pathogenic bacteria¹¹.
qPCR works by amplifying the DNA of a pathogenic strain suspected to be present. The amount of DNA is then measured to determine if the bacteria is present in harmful amounts¹¹. Because of this, qPCR is specific and can’t be generalized across multiple bacteria types. It’s only used when a specific strain is suspected of causing an infection.
Transcriptomics, Proteomics & Metabolomics
Beyond qPCR, there are a few other diagnostic tests to assess gut health, including transcriptomics, proteomics, and metabolomics.
Transcriptomics reveals active gene expression in microbiomes, showing which genes are actively used to sustain bacteria. Unlike shotgun or 16S sequencing, which show the DNA present, transcriptomics highlights genes that are currently active and contributing to physiological processes¹¹.
Proteomics focuses on identifying the proteins in our gut and their functions. It helps understand interactions between bacteria, which is crucial for diagnosing diseases influenced by bacterial proteins¹¹.
Metabolomics analyzes metabolic products like sugars, carbohydrates, lipids, and organic acids to reflect the functional state of the microbiome. Many diseases caused by pathogenic bacteria are linked to these molecules, so analyzing their levels can help diagnose infections and guide treatment¹¹.
The Future of Microbiome Testing
Scientists are working on exciting new sequencing methods that can read entire DNA strands without needing to break them into smaller fragments. This advancement promises to make sequencing even more efficient and high-throughput, speeding up the process and reducing costs by eliminating the need for DNA fragmentation¹⁰.
These cutting-edge technologies aim to study DNA at microscopic scales, eliminating the need to synthesize extra DNA strands for a macroscopic sample. Achieving this level of precision and efficiency involves innovative techniques that can manipulate and analyze DNA at the microscopic level¹⁰.
SMRT sequencing uses a process where a DNA strand is illuminated with light simultaneously as individual fluorescent nucleotide “tags” are added. A computer then records these microscopic events and translates them into a complete genome sequence¹⁰.
Nanopore sequencing works by guiding a single tiny DNA molecule through a minuscule biological pore with the help of electrical currents. When different nucleotides move through this nanopore, they create a distinct charge that a computer detects and translates into genetic information¹⁰.
These novel approaches promise to revolutionize DNA research and analysis by offering higher accuracy and reduced experimental complexity. While these technologies are promising, they still have a long way to go before they can accurately, efficiently, and cost-effectively detect our microbiome.
While technology continues to evolve as scientists push the boundaries of the field, interest in gut microbiomes remains strong. Embrace the journey of discovering the fascinating world within you – gut health insights are just a test away!
What is the Best Sequencing Method For The Microbiome?
The reliability, cost-effectiveness, and high-throughput nature of targeted 16S sequencing make it the go-to method for microbiome profiling. This technique has been widely and successfully used in research linking diseases to dysbiosis – imbalances in the microbial community – and can help give you more information about your gut too.
GUTXY’s own microbiome testing kits use next-generation sequencing (NGS) methods to provide detailed reports on your gut microbiome—give it a try and see what’s inside your gut!
These testing kits work by collecting stool samples and preserving them in a tube filled with a special solution that stabilizes the sample during transit to the lab. This ensures the sample can be properly analyzed upon arrival. It’s a convenient way to get meaningful insights about your gut health right from the comfort of your home.
Microbiome testing has become a game-changer in healthcare, shedding light on how our gut bacteria impact weight, mental health, and disease. Recent studies have uncovered links between specific gut compositions and conditions like obesity and autoimmune disorders, guiding more targeted treatments.
With advanced techniques like metagenomic sequencing, we’re delving deeper into the microbiome’s mysteries, revolutionizing personalized healthcare approaches. So, as we continue to unlock the secrets of the microbiome, the future of personalized health looks brighter than ever before!
