ABOVE: Rare bacterial strains have been difficult to capture when sequencing the human microbiome. ©istock, fatido

Human bodies are teeming with trillions of microbial cells that comprise the microbiome, many of them bacteria. Although they may be small, some of these bacteria maintain health, but others promote sickness. These differences often come down to the genes in each bacterial genome, but it can be challenging to find and sequence rare strains.

Headshot of Gang Fang, a geneticist at the Icahn School of Medicine at Mount Sinai, holding a book in his laboratory.
Gang Fang’s team developed mEnrich-seq, a sequencing technique that enriches bacteria of interest in a sample.
Brian Schutza

Gang Fang, a geneticist at the Icahn School of Medicine at Mount Sinai, has proposed a new solution called mEnrich-seq that draws on his decade of bacterial epigenomic research to distinguish different species’ DNA for metagenomic studies.1 In an interview with The Scientist, Fang describes his vision for how mEnrich-seq can help scientists answer hard questions about humans' bacterial companions.

What are some challenges associated with studying the human microbiome with metagenomics?

We have a lot of technologies to understand the microbiome in many different ways, but there is a common problem. If a bacterial species is abundant in a sample, then we can learn almost anything about it, but if the abundance of a species is really low, it is very hard to study. There may even be two or three coexisting strains of the same species, and the important strain may not be the one that is relatively more abundant. These different strains are often very similar in terms of their genomes, so it is extremely hard to differentiate between them.

What motivated you to develop mEnrich-seq?

If a target is rare, most of the sequencing throughput will be consumed by the more abundant species. The moment we sequence, we have already lost this battle, so we needed a new strategy before sequencing. 
– Gang Fang, Icahn School of Medicine at Mount Sinai

If a target is rare, most of the sequencing throughput will be consumed by the more abundant species. The moment we sequence, we have already lost this battle, so we needed a new strategy before sequencing. The natural epigenetic barcodes in bacteria give us a unique way to solve this problem. Even though different species and strains have similar genomes, they often encode different DNA methyltransferases, which determine their DNA methylation patterns.2 Bacteria do this to differentiate between self and foreign DNA. We can use this to differentiate between species’ or strains’ genomes based on the global methylation pattern. 

If we want to target a certain genome and we know its methylation pattern, we can rationally choose restriction enzymes that will cut at a certain sequence called the methylation motif. The enzymes will digest the vast majority of the background DNA that does not have this matching methylation. With mEnrich-seq, we can enrich bacteria of interest over 100-fold.

How does this protocol compare to a standard metagenomic sequencing experiment?

We actually considered this in our design. We wanted it to be effective but also very easy to plug into the existing pipeline. Ultimately, mEnrich-seq only involves two steps in addition to the standard library preparation. In the first step, after adapter ligation and before amplification, we digest the DNA using the rationally chosen restriction enzyme. By digesting the DNA with the adapter already ligated, only the intact DNA will have ligated adapters on both ends and can be amplified across their full length. The other DNA will be much shorter, which leads to step two: after amplification, we perform size selection. The other steps, such as quality control, remain the same. 

In what contexts do you think this method could be most useful?

One application is to battle antibiotic resistance, for example in urinary tract infections (UTI). Ideally, clinicians want to sensitively detect the antibiotic-resistance genes carried by a patient’s UTI strain, but a urine sample has a lot of host DNA and other bacteria. Right now, the best practice for UTI is to culture the urine samples, which takes three days to get a result. This is not ideal. We want to build an antibiotic resistance profile quickly—within one day—so that the doctor can decide which antibiotics to give a patient. 

Another application is for beneficial bacteria, or probiotics, such as Bifidobacterium. Different strains can have very different health benefits. If we want to discover probiotics associated with human disease or drug responses, we need to do some initial screening to narrow down the species in fecal samples and recover more promising candidates. 

A lot of people are interested in these applications, and we think mEnrich-seq provides a new, more sensitive, reliable, and cost-effective way to tackle these problems.

This interview has been condensed and edited for clarity.

References

  1. Cao L, et al. mEnrich-seq: methylation-guided enrichment sequencing of bacterial taxa of interest from microbiome. Nat Methods. 2024;21(2):236-246.
  2. Beaulaurier J, et al. Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation. Nat Biotechnol. 2018;36(1):61-69.