scMAR-Seq: a novel workflow for targeted single-cell genomics of microorganisms using radioactive labeling

Current methods for the identification of specific microorganisms based on an in situ metabolism are often hampered by insufficient sensitivity and habitat complexity. Here, we present a novel approach for identifying and sequencing single microbial cells metabolizing a specific organic compound with high sensitivity and without prior knowledge of the microbial community. The workflow consists of labeling individual cells with a [$^{14}$C] substrate based on their metabolic activity, followed by encapsulating cells in alginate with nuclear emulsion by using microfluidics. We here adapted the concept of microautoradiography to visually distinguish between encapsulated labeled and non-labeled cells, which are then sorted via flow cytometry for single cell genomics. As a proof-of-concept, we labeled, separated, lysed, and sequenced single cells of the benzene degrader Pseudomonas veronii from mock microbial communities. The cells of P. veronii were isolated with 100% specificity. Single-cell microautoradiography and genome sequencing is an innovative method for elucidating microbial identity, activity, and function in diverse habitats, contributing to elucidate novel taxa and genes with potential for biotechnological applications such as bioremediation.