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New Bacterial Epigenetic Sequencing Method Could Be Boon for Complex Microbiome Analyses


NEW YORK – A new sequencing method that exploits bacterial epigenomic patterns hints at ways to identify and sequence extremely low-abundance microbes and those challenging to culture in a cost- and time-effective manner.

The method, called mEnrich-seq, was pioneered by researchers at the Icahn School of Medicine at Mount Sinai and published yesterday in the journal Nature Methods.

Most microbiome studies to date assess microbial diversity, for which well-established techniques such as metagenomic shotgun sequencing are readily available and affordable.

Gang Fang, a professor of genetics and genomic sciences at Mount Sinai and the senior author of the study, said that mEnrich-seq will enable researchers to more effectively zero in on individual components of the microbiome, particularly with respect to species that are of low abundance or that are challenging to cultivate.

Assessing diversity, Fang said, will remain relevant in cases such as evaluating the effects of antibiotics on a person's microbiome, as this is known to shift diversity patterns.

"The target user and target applications [for mEnrich-seq]," Fang said, "are those that would really benefit from a high-resolution, reliable genome of pathogens, beneficial bacteria, or low-abundance but important species."

Fang made an example of Akkermansia muciniphila, a Gram-negative bacterium colonizing the intestinal tract that his lab has been studying and which is implicated in numerous conditions and disorders, including obesity and type 2 diabetes, as well as patient responses to cancer immunotherapies.

"Akkermansia is very hard to culture," Fang said. "It would take weeks for you to culture it, and you need special equipment, special expertise. It's very tedious."

Even some common pathogens such as Escherichia coli, Fang said, can take days to culture and test for antibiotic resistance or sensitivity, which could impact patient care. 

Fang described mEnrich-seq as a "tweezer" capable of selectively isolating and sequencing the DNA of these target organisms by their DNA methylation patterns.

Bacteria have three major forms of DNA methylation: N6-methyladenine (6mA), N4-methylcytosine (4mC), and 5mC, all catalyzed by methyltransferases that add methyl groups to DNA in a highly sequence-specific manner. Furthermore, all genetic components such as chromosomes and plasmids in a given bacterial strain share the same set of methylation motifs, and most methylation sites are highly stable. Finally, the methylation motifs are highly variable between different species and even different strains of the same species.

In his group's study, Fang and his colleagues demonstrated mEnrich-seq's utility in several contexts. They first selectively sequenced E. coli genomes from urine samples derived from patients with urinary tract infections, comparing these results to metagenomic shotgun sequencing and to selective sequencing of E. coli isolates cultured from the same samples.

They next selectively sequenced A. muciniphila genomes from three infant fecal samples, showing that mEnrich-seq successfully covered approximately 99.7 percent of that organism's genome.

The group then explored their method's ability to enrich and sequence low-abundance species. Since less is known about these species, particularly with respect to methylation motifs, Fang's group first had to identify these patterns using existing methylation discovery tools from microbiome samples. Although this method was more time-consuming and included an initial round of shallow sequencing, the researchers successfully isolated and sequenced several low-abundance species, such as Alistipes finegoldii, Bifidobacterium longum, Dorea longicatena, and Barnesiella intestinihominis.

Jack Gilbert, a professor of pediatrics at the University of California School of Medicine and director of the Microbiome and Metagenomics Center, called mEnrich-seq "innovative and potentially impactful" and said that it employs a unique approach of exploiting bacterial DNA methylation patterns to enhance metagenomic sequencing efficiency by enriching targeted bacterial taxa in microbiome samples.

"By enabling selective sequencing of specific bacterial taxa, mEnrich-seq could significantly enhance our ability to study and understand complex microbiomes," he said. "I, for one, would like to explore this not just in stool to isolate particular strains of specific taxa but also in natural environments to help with identifying key organisms in complex systems like soil and sediment."

Gilbert noted that while the different parts of Fang's study are not entirely novel in themselves, bringing together these methodologies and showing, in particular, that if you have the right info on methylation that you can use it to isolate particular groups of bugs "is cool."

Nonetheless, Gilbert cautioned that a potential weakness could be mEnrich-seq's reliance on known methylation patterns, which could limit its utility for unknown or highly variable taxa. This, he said, could be a particular problem for poorly characterized systems such as the soil microorganisms that interest him.

"The study thoroughly evaluates the method, but further real-world applications would be beneficial to establish its reliability fully," Gilbert said. "One might want to know how it performs across a broader range of microbiome samples and its efficacy in detecting novel or highly variable bacterial taxa."

Fang expects that the method's broad applicability will make it useful in both research and diagnostic settings. Despite the likely need for extra work to isolate and enrich low-abundance species, Fang said that most known pathogenic bacteria have certain known conserved methylation patterns that make them ideal targets for mEnrich-seq.

"Every single strain of E. coli has two conserved methylation patterns, for example" Fang said. This conservation is seen in other pathogens, including Salmonella, Pneumoniae, Chlamydia, and Akkermansia.

A patent is currently pending for mEnrich-seq, and Fang said that he is "actively discussing" business opportunities in the diagnostics field with several unspecified companies.

Fang also plans to use mEnrich-seq in more studies over the new year. While continuing his studies on Akkermansia, Fang also intends to investigate at least one other medically relevant microbe.

"I may not name that bacteria yet," he said. "Basically it is another bacteria that will also greatly benefit from this strategy. Super hard to culture, but a great medical impact if we know its genome."