Fast Encapsulation of Microbes into Dissolvable Hydrogel Beads Enables High‐Throughput Microbial Single‐Cell RNA Sequencing of Clinical Microbiome Samples

Microbial single-cell RNA-seq (mscRNA-seq) can achieve resolution at the cellular level, enhancing the understanding of microbial communities. However, current high-throughput mscRNA-seq methods are limited by multiple centrifugation steps, which can lead to microbial loss and bias. smGel-seq is reported, a high-throughput single-microbe RNA sequencing method for clinical microbiome samples that employs hydrogel beads to encapsulate individual microbes to reduce microbial loss and input requirements. In this method, a novel microchannel array device is implemented for encapsulating single microbe in dissolvable hydrogel beads (smDHBs), along with an optimized automated microfluidic platform to co-encapsulate barcoded beads and smDHBs, enabling high-throughput barcoding of individual microbes. smGel-seq significantly increases the microbial recovery rate in a gut microbiome sample from 8.8% to 91.8%. Furthermore, this method successfully processes clinical microbiome samples with microbial inputs 20 times lower than those required by previous methods. Notably, smGel-seq enables the first mscRNA-seq in a clinical sputum microbiome sample, revealing a specific microbial subpopulation that may play a key role in environmental adaptability, antibiotic resistance, and pathogenicity. These results highlight the compatibility of smGel-seq with clinical microbiome samples and demonstrate its potential for widespread application in diverse clinical and research settings.