Presented by: Ya Lea Wang
Characterization of microbial community viability is of great importance: essentially all sequence-based technologies do not differentiate living from dead microbes, whereas the functions and phenotypes of microbial communities (including the human microbiome) are defined by biochemically active (“viable”) organisms. As a result, our understanding of microbial community structures and their potential mechanisms of transmission between humans and our surrounding environments remains incomplete. As a potential solution to this issue, 16S RNA-based amplicon sequencing (not rRNA gene, i.e. 16S-associated DNA) has been proposed as a method to quantify the viable fraction of a microbial community, but its reliability has not been evaluated systematically.
Here, we present our work to benchmark 16S-RNA-seq (targeting 16S rRNA transcripts and genes for parallel RNA and DNA sequencing) for viability assessment in synthetic and realistic microbial communities. In synthetic communities, we found that 16S-RNA-seq successfully reconstructed the mixtures of heat-killed Escherichia coli and Streptococcus sanguinis. We further applied this technique to swabbings of natural microbial communities (computer screens and mice, soil, and saliva) spiked with known concentrations of living and heat-killed E. coli to evaluate its performance under variable biomass, chemical background and diversity conditions. No significant compositional differences were explained by the 16S-RNA assessment, suggesting that 16S-RNA-seq is not appropriate for viability assessment in complex communities. Results were slightly different in our validation using environmental samples of similar origins (i.e. from Boston subway systems), samples were differentiated both by environment type as well as by library type, though compositional dissimilarities between DNA and RNA samples remained low. Overall, these results show that 16S-RNA-seq has promise, but that previous literature assuming that 16S-rRNA amplicons are directly, quantitatively enriched for viable microbes is likely incorrect. Potentially due to the stability of this uniquely non-protein-coding RNA or its abundance being highly non-correlated with underlying microbial cell count, 16S rRNA amplicons do not reflect microbial viability outside of very simple, synthetic “communities.”
We are currently continuing this work to develop new RNA amplicon-seq markers based on protein-encoding genes such as rpoB and cpn60, as well as to improve viability assessment with multi-omic integration, e.g. combining amplicon-seq with functional indicators such as metatranscriptomic and metaproteomic profiles. These can circumvent some of the current limitations of 16S-RNA-seq, providing a complementary definition of viability, and directly observing activities such as virulence, pathogenicity, or antimicrobial resistance that are not captured by amplicon sequencing.
Ya Lea Wang – Poster Description (Audio Clip)
If you have any questions regarding the poster, feel free to reach out to Ya Lea Wang here.