A new advanced, high-throughput sequencing technique should enable scientists to have a firmer grasp on the activity of naturally occurring microbiomes in response to real-world conditions and diet. Investigators at the University of Chicago developed the new technique to directly analyze transfer RNA (tRNA)—providing a clearer picture of microbial communities’ responses to various environmental changes, such as varying temperatures or the changing availability of nutrients. Findings from the new study were released today in Nature Communications through an article titled “Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis.”

In the current study, the research team demonstrated the application of tRNA sequencing to gut microbiome samples from mice that were fed either a low-fat or high-fat diet. Using new software and computational strategy described in the study created a catalog of tRNA molecules recovered from the gut samples, traced them back to the bacteria responsible for their expression, and measured chemical modifications in tRNA that take place after transcription.

“We report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts,” the authors write. “Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.”

Each tRNA in bacteria has an average of eight chemical modifications that can tune its function. The new high-throughput sequencing and analysis strategy detects two of them, but it can also measure the amount of modification on a scale from 0 to 100 percent at each site. The level of one of the modifications, called m1A, was higher in the gut microbiome of mice that were fed a high-fat diet. This is the first time scientists have been able to see any modification level change in tRNA in any microbiome.

“We were working backward,” notes senior study investigator Tao Pan, Ph.D., professor of biochemistry and molecular biology, at the University of Chicago. “We had no preconceived notion of why the m1A tRNA modifications were actually there or what they were doing, but to see any modification change at all in the microbiome is unprecedented.”

The m1A modification helps synthesize certain types of proteins that may be more abundant in a high-fat diet. The researchers don’t know yet if these modification differences occur in response to that diet, or if they are already present and become active to enhance the synthesis of those proteins.

This study is the first of a series of microbiome projects from the University of Chicago funded by a grant from the Keck Foundation. Dr. Pan has pioneered the use of tRNA sequencing tools, and the grant will fund continuing work to make them widely accessible through new computational strategies that co-senior study investigator A. Murat Eren, Ph.D., assistant professor of medicine at the University of Chicago, develops. Large sets of data generated by tRNA sequencing can provide critical insights into microbiomes associated with humans or the environment at a low cost.

“The molecular and computational advances that have emerged during the last two decades have only helped us scratch the surface of microbial life and their influence on their surroundings,” Dr. Eren states. “By providing quick and affordable insights into the core of the translational machinery, tRNA sequencing may become not only a way to gain insights into microbial responses to subtle environmental changes that can’t be easily measured by other means, but also bring more RNA biology and RNA epigenetics into the rapidly developing field of the microbiome.”

The researchers were excited by their findings but agree there is much room to improve this novel strategy, and they hope that it will happen quickly.

“There are a number of ways to examine microbiome activities, but nothing is faster and gets you more volume of data than sequencing,” Dr. Pan concludes. “Here we have developed a new method that reports activity of the microbiome through tRNA and does so at high throughput. That’s really the value.”