Revolutionizing Microbial Single-Cell Sequencing: DoTA-seq Unveils a Simple Yet Powerful Method 

Photo credit: Qin Dai

In a recent study, a research team led by Dr. Freeman Lan has developed a method for single-cell genetic profiling of microbes. The findings, published in Nature Methods, introduce a robust and easily adaptable droplet microfluidics workflow named DoTA-seq (Droplet Microfluidics for Targeted Amplification Sequencing), providing a scalable solution for studying single-cell heterogeneity in microbial populations. 

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

The authors 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. 

Enter DoTA-seq, a method that leverages droplet microfluidics—a platform technology in which reactions occur in picoliter-scale droplets manipulated through microfluidic channels. Unlike previous droplet microfluidics-based methods, DoTA-seq uses simple modules like microfluidic droplet makers and gel bead re-injectors, eliminating the need for complex microfluidics expertise. This makes DoTA-seq suitable for academic laboratories with moderate budgets. 

DoTA-seq’s targeted approach allows for high capture rates of loci of interest, making it ideal for single-cell sequencing assays with known target regions. This innovation, which exploits ultrahigh-throughput droplet microfluidics, showcases simplicity, efficiency, and potential for a microfluidics-free adaptation in the future. 

The research team successfully applied DoTA-seq to various microbial populations, demonstrating 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. 

Dr. Freeman Lan, the lead author of the study, expressed excitement about the potential applications of DoTA-seq. “Our method opens new avenues for exploring microbial diversity and functions at the single-cell level. DoTA-seq’s simplicity and broad applicability make it an invaluable tool for the biology research community. We are particularly excited to apply this technology to track the spread of antibiotic resistance genes in the future.” said Dr. Freeman Lan. 

The study marks a significant step forward in microbial single-cell sequencing, offering a promising avenue for researchers to unravel the intricacies of bacterial populations.