Most of our knowledge about bacterial functional roles in microbiomes comes from bulk measurements. Yet microbial communities are complex ecosystems in which functionally distinct bacterial subpopulations with unique transcriptional states emerge across environmental niches and from interactions with other community members. Such heterogeneous transcriptional states are inherently missed by bulk measurements. To address this gap, we developed multispecies microbial split-pool ligation meta-transcriptomics (metaSPLiT), a scalable, instrument-free single-cell RNA sequencing approach for the microbiome. Using metaSPLiT, we profiled healthy human fecal microbiomes and reconstructed 21,598 single cell transcriptomes belonging to 70 unique bacterial species.

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Virtually every surface of our planet is colonized by microbes and the human body is no exception. The 38 trillion microbes living in our gut metabolize a large fraction of the dietary metabolites, pharmaceutical drugs, and xenobiotics we consume and thus provide an important interface between the environment and our blood stream.

Whereas many of the species forming the human gut microbiome have been well-characterized by now, we still know very little about the factors that determine which microbial species can coexist in any given individual. This currently limits our understanding regarding engraftment of pathogens and probiotics and how those will interact with other microbes and host tissues in the human gut.