Sequencing the Human Microbiome: Opening the Black Box

The field of microbiome research has evolved at a tremendous pace in recent decades, with mounting evidence that our microbial communities have a profound impact on our health.

“To put it into context, we have a ballpark figure that the microbiome is about 150 times bigger than the human genome,” says Lindsay Hall, Microbiome Group Leader at the Quadram Institute, Norwich, UK. “So that begins to give a bit of an idea that it might be quite important for us!”

Huge advances in sequencing technologies, paired with dramatic decreases in costs, have opened the door to studies exploring the collection of microbes that live on and within our bodies more comprehensively than ever before.

“We’ve known for a long time that these communities are important for our health but trying to get a handle on their complexity was very difficult,” explains Hall. “But we can now drill down and have a look at these microbes and see what impact they have on our health.”

Improving our knowledge of how the composition and function of our microbiome can influence aspects of our physiology holds great promise for the development of new ways to prevent and treat a range of medical conditions - such as infectious diseases, cancer, mental health disorders and autoimmune conditions.

Sequencing the "dark matter"

Although microbiome research is by no means new, for many years’ scientists were limited by the traditional microbiology methods that were available to study it.

“The problem is that it is not possible to culture much of the microbiota under standard conditions,” explains W. Florian Fricke, Professor of Nutrigenomics at the University of Hohenheim, Stuttgart, Germany. “So for a long time, that part was overlooked.”

But around a decade ago, the introduction of next-generation sequencing (NGS) technologies accelerated research into this hidden fraction of the microbiome.

“We can now actually analyze microbial communities without having to grow them in the lab,” explains Alex Almeida, Postdoctoral Research Fellow at the European Bioinformatics Institute (EMBL-EBI) in Cambridge, UK. “This has allowed us to examine the diversity of the microbiome to a much greater depth.”

Researchers use two main NGS approaches to analyze the microbiome. Metagenomics involves sequencing all of the DNA within a sample, while amplicon sequencing looks at specific bacterial "fingerprints" – typically by amplifying and sequencing fragments of the 16S rRNA gene.

“Metagenomics is more expensive but provides a very deep, complete picture of the microbial community in a sample – so not just the bacteria, but also other organisms such as viruses too,” explains Fricke. “Amplicon sequencing is simpler and quicker and can tell you what bacterial species are present and their relative abundance.”

Metagenomics produces a collection of short DNA fragments that scientists then try to piece together into individual bacterial genomes.

“When you sequence a complex community like the human gut, you get lots of different bits and pieces from many different species,” explains Almeida. “On the computational side, we need to disentangle this diversity to try to tease apart the individual organisms.”

But newer technologies can generate much longer sequences, enabling much greater resolution and accuracy. And chromosomal conformation capture techniques, such as Hi-C, can provide clues about the real-life proximity of two fragments within the sample.

“If we know that two DNA molecules were closer together, we have a greater assumption that they belong to the same organism and this improves the analyses,” explains Almeida. “These techniques allow us to add another dimension to the sequencing data that we can retrieve.”

Ongoing challenges

NGS technologies combined with bioinformatics has led to an explosion of studies into the microbiome in laboratories around the world.

“Thousands and thousands of samples are being sequenced and released to the public domain every month,” says Almeida. “The ability to analyze them consistently and reproduce the results that are done by different teams is a big challenge in the field.”

To help tackle this issue, Almeida’s team at the EBI is responsible for MGnify, a centralized hub for microbiome-derived sequencing data, which as he explains is, “a free resource that enables researchers to deposit and analyze their data in a consistent manner.”

Although the advent of advanced sequencing technologies has opened the door to exploring the microbiome in greater depth than ever before, several aspects of microbial communities remain inaccessible.

“We still get a lot of ‘unknown’ sequences – so we might know that it’s bacterial, but we don’t know what species it is,” says Hall. “Increasing reference databases will help, but this will require a lot more culturing.”

Another limitation is that current methodologies often lead to the generation of compositional data rather than absolute quantitative information about the microbes within a sample.

“You don’t usually keep the information of the original quantity of the microbiome that you analyzed, so in the end, you only get a percentage of an unknown total,” explains Fricke. “So we may find that 20% is bacterium X and 30% is bacterium Y – but we just end up with a relative abundance species catalog.”

To get a handle on the full complexity of the microbiome, researchers will need to develop new approaches that enable them to uncover the quantitative relationships between major microbial groups.

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Huge progress in microbiome analysis

Despite these challenges, researchers are making incredible progress in building our understanding of the microbiome. For example, Almeida was part of a team that recently published their results from a large-scale sequencing project that involved reconstructing 92,143 metagenome-assembled gut genomes from 11,850 people.

“We now have a much more comprehensive map of the human gut microbiome,” describes Almeida, “We found almost 2,000 previously unknown bacterial species, substantially expanding the known repertoire of the collective gut microbiota.”

But the development of miniaturized devices is the latest paradigm shift in sequencing technology - opening exciting new opportunities for researchers to develop pioneering solutions to tackle real-world problems.

“There are new devices now available that are around the size of a USB stick,” says Almeida. “These technologies are making it possible to collect samples in the field and analyze them in real-time rather than sending them back to the laboratory.”

As proof-of-principle of what these cutting-edge technologies could offer in a clinical setting, Hall’s team recently showed that it is possible to carry out metagenomics to rapidly and reliably identify the microbes present in a preterm baby’s stool that may cause life-threatening conditions.

“We used a sequencing device coupled with bespoke software to analyze the data in less than five hours,” explains Hall. “Our method also uncovers the presence of antimicrobial resistance genes, which is vital information for doctors to select what antibiotics are going to be the most effective at killing it.”

"Cautious optimism"

Thanks to huge advances in sequencing technologies and its computational analysis, our understanding of the microbial communities that inhabit the human body has greatly improved in recent decades.

“We’ve got the most densely colonized ecosystem on the planet living in us which is a bit mind-boggling!” says Hall. “So it’s very important to know who they are and what they do for us – and these new technologies are enabling us to figure out what’s there or what they’re doing.”

Using that information has tantalizing opportunities for the development of new medical interventions that could transform our approach to preventing or treating many different diseases.

“I’m very, very excited about the potential of the microbiome,” says Almeida. “It could be revolutionary, although we have to be cognizant that there is a lot of progress that still needs to be made.”