Microbiome Sequencing: A Bigger Picture of Smaller Things

We’ve known about bacteria and other microorganisms for centuries and still we know surprisingly little about the almost unfathomable diversity of microscopic life around us – or even on and inside us. Why is this and how can next generation sequencing make a difference?

Since the late 1970s, it has been possible to find a genetic code from directly studying strands of DNA. These methods, the most common being Sanger sequencing, gave us the first human genome in 2003. Since then, advanced next generation sequencing (NGS) has come of age, giving researchers orders-of-magnitude improvements in speed and cost. NGS has made it possible to look efficiently at some of the smaller creatures out there and try to sequence microbiomes.


What is a microbiome?

A person’s microbiome can be defined as the genomes of all microorganisms living in and around them put together. As the number of bacteria living in our gut alone is likely to be in the trillions, the amount of information contained in a microbiome is far greater than any human genome.

It is well known that the ‘health’ of our microbiome has a direct impact on our own health – and tips on improving gut health (as well as products) are easy to find. As a result, a lot of research is focused on figuring out what’s in our microbiome and how exactly it affects us. This information is important for many conditions, ranging from obesity and inflammatory bowel disease to autism spectrum disorder.


Investment in research

With increased understanding of the fact that microbes affect health in ways other than simply causing infections, interest in microbiome research began to spread outside of academia. The biotechnology and pharmaceutical industries began to see opportunities. If there’s a link between a person’s microbiome and a disease, might their microbiome also hold the key to treatment?

A good example of this increased interest is the Microbiome Coalition: a collaboration between a range of thought leaders and companies including AO Biome®, Abbott Nutrition, CosmosID®, Diversigen®, the Mayo Clinic®, Second Genome® and Whole Biome®. Launched in 2014, it aims to remove hurdles to commercial success and pave the way for approval of microbiome-based treatments and diagnostics.

Government funding also plays a key role in advancing the microbiome field. For example, in 2016 the US government, together with other stakeholders, launched the National Microbiome Initiative. Its scope is broader than human health alone as it looks at the microbiomes of whole ecosystems as well, and its findings could have important implications for environmental protection, food safety and even climate change.


NGS for microbiome sequencing

Although microbiology has come a long way since the days of Van Leeuwenhoek and Pasteur, much research still relies on microbe culture before analysis. This method is well suited for studying many individual microbes, but when looking at the diversity within a given sample it can introduce a substantial bias – one that favors microbes that are easy to culture.

Addressing this bias, molecular techniques – and NGS in particular – can use freshly extracted DNA without the need for culture. This method avoids overrepresentation of easy-to-culture species and gives an unbiased picture of genetic diversity. NGS technology can sequence the entire genome of a species, or it can zoom in on specific regions of the genome that are helpful for species identification (for bacteria and archaea, the 16S rRNA region is often used). This method enables faster and more efficient microbiome analysis.

Regardless of the exact NGS method, a key step is the preparation of a sequencing library. This involves turning freshly isolated nucleic acids into fully prepared, bitesize chunks that are ready for sequencing. This step may involve amplification, fragmentation, attachment of adapter sequences and size selection – and is often both time-consuming and labor-intensive.


The importance of standardization

One reason why NGS sample preparation and standardization is challenging is because of the high number of liquid handling operations – especially in applications that involve working with many different species, such as microbiome research. With more liquid handling the risk of errors also increases, which leads to outliers that affect the reliability of the entire study. Standardization helps to keep the error rate low as the size of an experiment goes up.

One way to ensure better standardized NGS libraries and also make the process less labor-intensive is to automate liquid handling. Repeated pipetting, mixing and separation are all well suited to automation by reducing errors and ensuring high accuracy, even at small sub-microliter volumes. As a result, the use of automated liquid handling technology improves both consistency and speed in NGS library preparation for reliable results.

Take a look at our successful Webinar: Towards Standardized Workflows - A focus on microbiome insights and learn how to standardize your microbiome research and NGS library preparation.


Eppendorf offers a wide product range to support different aspects of NGS workflows. It includes the epMotion 5073m and 5075t® which are easily integrated into many Illumina®, KAPA® and NEB® workflows – bringing accuracy, consistency and ease of use to liquid handling for high-quality NGS libraries.

Read our article on whole genome sequencing to get more information on how to optimize NGS with automation.

 

AOBiome® is a registered trademark of AOBiome, LLC, USA. CosmosID® is a registered trademark of CosmosID Inc., USA. Diversigen® is a registered trademark of Diversigen, Inc., USA. Mayo Clinic® is a registered trademark of Mayo Foundation for Medical Education and Research, USA. Second Genome® is a registered trademark of Second Genome Inc., USA. Whole Biome® is a registered trademark of Whole Biome, Inc., USA.

Eppendorf®, the Eppendorf Brand Design and epMotion® are registered trademarks of Eppendorf AG, Germany.