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Professor Freeman Lan (BME) and his team plan to expand the application of the DoTA-seq. (Photo: Submitted)

A research team led by Professor Freeman Lan (BME) has developed a method for single-cell genetic profiling of microbes.  

The study, which was published in Nature Methods, introduces a robust and easily adaptable droplet microfluidics workflow named Droplet Microfluidics for Targeted Amplification Sequencing (DoTA-seq), which provides a scalable solution for studying single-cell heterogeneity in microbial populations.  

“Our method opens new avenues for exploring microbial diversity and functions at the single-cell level,” says Lan. “DoTA-seq’s simplicity and broad applicability make it an invaluable tool for the biology research community.” 

Single-cell genetic heterogeneity is a pervasive phenomenon in bacteria, influencing critical processes such as evolution, antimicrobial resistance, host colonization and pathogenesis. Traditional methods, such as colony plating, fall short in capturing unculturable taxa and detecting rare variants. These methods cannot observe mechanisms driving heterogeneity operating on timescales similar to colony growth.  

The researchers highlight the difficulty of implementing existing single-cell sequencing methods for microbes due to species-specific protocol optimization challenges. Bacteria’s small size, low genetic material yield and diverse cell membrane compositions further complicate the development of generalizable sequencing workflows.  

The DoTA-seq method leverages droplet microfluidics — a platform technology in which reactions occur in picolitre-scale droplets manipulated through microfluidic channels.  

Unlike previous droplet microfluidics-based methods, DoTA-seq uses simple modules such as microfluidic droplet makers and gel bead re-injectors, which eliminate the need for complex microfluidics expertise. This makes DoTA-seq suitable for academic laboratories with moderate budgets, says Lan. 

DoTA-seq’s targeted approach allows for high capture rates of loci — the locations of specific genes on a chromosome, making it ideal for single-cell sequencing assays with known target regions. The innovation, which exploits ultrahigh-throughput droplet microfluidics, aims to offer a simple and efficient pathway towards microfluidics-free adaptation in the future.  

The research team applied DoTA-seq to various microbial populations to demonstrate its versatility. In one assay, they tracked shifts in the prevalence of antibiotic-resistance genes in a human gut microbial community exposed to increasing antibiotic concentrations.  

Another assay revealed taxonomic associations of antibiotic-resistance genes and plasmids in mouse and human fecal samples, respectively. Lastly, the team quantified genetically distinct subpopulations resulting from phase variation in the human gut symbiont Bacteroides fragilis.   

Looking forward, Lan and his team plan to expand the application of DoTA-seq. 

“We are particularly excited to apply this technology to track the spread of antibiotic resistance genes in the future,” he says. 

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