Viable bacteria-targeted single-cell genome sequencing

Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities in microbiome analyses. However, signals derived from non-viable bacteria and non-cell DNA may inhibit the characterization of the communities. Here, we present a method for viable bacteria-targeted single-cell genome sequencing to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities in order to determine the bacterial survival profile at the species or strain level. A massively parallel single bacterial genome sequencing technology called PMA-SAG-gel uses gel matrixes that enable sequential enzymatic reactions for cell lysis and genome amplification of viable single cells from the microbial communities. PMA-SAG-gel removed the SAGs derived from dead bacteria and enabled selective sequencing of viable bacteria in the model samples of Escherichia coli and Bacillus subtilis. Next, we demonstrated the recovery of near-complete single-amplified genomes (SAGs) of four oxygen-tolerant Bacteroides spp. and Phoieicola spp. from fresh feces. Furthermore, we found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. PMA-SAG-gel enabled single-cell genome sequencing of surviving uncultured bacteria and identified specific viable bacterial strains in feces samples processed under aerobic condition. The survival profile of an entire population at the species or strain level will provide the information for understanding the characteristics of the surviving bacteria under the specific environments or sample processing and insights for quality assessment of live bacterial products or fecal microbiota transplantation and for understanding the effect of antimicrobial treatments.