The human gut microbiome is complex and diverse, with significant implications for health and disease. The study develops smRandom-seq2, a droplet-based method that overcomes the limitations of existing techniques by capturing RNA from a wide variety of species. The method uses optimized random primers and a triple-module computational pipeline to analyze bacterial and phage activities. smRandom-seq2 was applied to four fecal samples, identifying 29,742 single microbes and 329 unique species. The study reveals distinct adaptive states in Prevotella and Roseburia genera and intrinsic adaptive strategies in Phascolarctobacterium succinatutens. It also identifies novel host-phage transcriptional activity associations.
Key findings from the study include:

1. Development of smRandom-seq2: The study introduces smRandom-seq2, a high-throughput and high-resolution single-microbe RNA sequencing method. This method uses optimized random primers and a triple-module computational pipeline to analyze bacterial and phage activities in complex microbial communities. The technique captures RNA from a wide variety of species, making it highly adaptable to real-world samples.
2. Adaptive States in Prevotella and Roseburia: The study identifies distinct adaptive response states among species in the Prevotella and Roseburia genera. In Prevotella, species like P. copri and P. sp900767615 show significant differences in gene expression related to adaptive cellular responses. In Roseburia, species like R. hominis and R. intestinalis exhibit functional differences in cell motility and multidrug resistance.
3. Intra-population Heterogeneity in Phascolarctobacterium succinatutens: The study reveals intra-population functional heterogeneity in P. succinatutens. Three major subpopulations were identified, each showing distinct transcriptional states. Subpopulation 1 exhibits higher expression of genes related to mobile genetic elements and multidrug resistance, while subpopulation 2 shows increased activity in succinate metabolism pathways.
4. Host-Phage Activity Associations: The study identifies hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Using the MIC-Phage module, the researchers mapped phage sequences to bacterial genomes, revealing the specific interactions between bacteria and their associated phages. These associations provide insights into the regulatory roles of bacteriophages in the gut microbiome.

smRandom-seq2 is a powerful and scalable technique that provides a comprehensive understanding of the human gut microbiome. It captures the transcriptional activities of individual microbes, revealing adaptive states and host-phage interactions. The method's high throughput and resolution make it a valuable tool for studying microbial communities in various contexts, from basic research to clinical applications. The work entitled “ High-throughput single-microbe RNA sequencing reveals adaptive state heterogeneity and host-phage activity associations in human gut microbiome” was published on Protein & Cell (published on May. 23, 2024).
DOI: 10.1093/procel/pwae027