Abstract
Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. However, signals derived from non-viable bacteria and non-cell DNA may inhibit its characterization. Here, we present a method for viable bacteria-targeted single-cell genome sequencing, called PMA-SAG-gel, to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities. 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 single-amplified genomes (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 SAGs of eight oxygen-tolerant bacteria, including Bacteroides spp. and Phocaeicola spp., from 1331 human feces SAGs. We found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. The survival profile of an entire population at the 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.
Highlights
Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities
Heat-treated E. coli or feces suspended in guanidine thiocyanate (GuSCN) solution were used as model samples
Since the sequencing results were obtained with a sixfold coverage, the 20% completeness was reasonable (Fig. 3b,c). These results indicated that propidium monoazide (PMA)-single-amplified genomes (SAGs)-gel could remove the SAGs derived from dead bacteria and enable the selective sequencing of viable bacteria
Summary
Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. One approach for characterizing viable bacteria from the microbial community is an assay combined with a chemical treatment for the removal of amplification signals derived from extracellular or non-viable cell DNA. Only DNA from viable cells with intact cell membranes will be amplified and detected as a signal from analyzed microbial communities This treatment has been used in combination with the polymerase chain reaction (PCR)[9,10], amplicon (16S rRNA gene) s equencing[2–4,11], and shotgun metagenomics[12,13] to selectively detect viable bacteria in various microbial communities and to determine the relationship between the diversity of viable bacteria and environmental conditions. In combination with PMA treatment, metagenomics approaches, including 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing, can provide comprehensive insight into microbial composition and function of viable bacterial cell populations. An ideal approach for the classification and functional characterization of uncultured viable bacteria would be to obtain their wholegenome sequences by targeting viable bacteria from complex microbial communities
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