Nanopore- and AI-empowered metagenomic viability inference
Authors: Urel Harika , Benassou Sabrina , Reska Tim , Marti Hanna , Rayo Enrique , Martin Edward J. , Schloter Michael , Ferguson James M. , Kesselheim Stefan , Borel Nicole , Urban Lara 
Published: June 2024

AbstractThe ability to differentiate between viable and dead microorganisms in metagenomic samples is crucial for various microbial inferences, ranging from assessing ecosystem functions of environmental microbiomes to inferring the virulence of potential pathogens. While established viability-resolved metagenomic approaches are labor-intensive as well as biased and lacking in sensitivity, we here introduce a new fully computational framework that leverages nanopore sequencing technology to assess microbial viability directly from freely available nanopore signal data. Our approach utilizes deep neural networks to learn features from such raw nanopore signal data that can distinguish DNA from viable and dead microorganisms in a controlled experimental setting. The application of explainable AI tools then allows us to robustly pinpoint the signal patterns in the nanopore raw data that allow the model to make viability predictions at high accuracy. Using the model predictions as well as efficient explainable AI-based rules, we show that our framework can be leveraged in a real-world application to estimate the viability of pathogenicChlamydia, where traditional culture-based methods suffer from inherently high false negative rates. This application shows that our viability model captures predictive patterns in the nanopore signal that can in principle be utilized to predict viability across taxonomic boundaries and indendent of the killing method used to induce bacterial cell death. While the generalizability of our computational framework needs to be assessed in more detail, we here demonstrate for the first time the potential of analyzing freely available nanopore signal data to infer the viability of microorganisms, with many applications in environmental, veterinary, and clinical settings.Author summaryMetagenomics investigates the entirety of DNA isolated from an environment or a sample to holistically understand microbial diversity in terms of known and newly discovered microorganisms and their ecosystem functions. Unlike traditional culturing of microorganisms, metagenomics is not able to differentiate between viable and dead microorganisms since DNA might readily persist under different environmental circumstances. The viability of microorganisms is, however, of importance when making inferences about a microorganism’s metabolic potential, a pathogen’s virulence, or an entire microbiome’s impact on its environment. As existing viability-resolved metagenomic approaches are labor-intensive, expensive, and lack sensitivity, we here investigate our hypothesis if freely available nanopore sequencing signal data, which captures DNA molecule information beyond the DNA sequence, might be leveraged to infer such viability. This hypothesis assumes that DNA from dead microorganisms accumulates certain damage signatures that reflect microbial viability and can be read from nanopore signal data using fully computational frameworks. We here show first evidence that such a computational framework might be feasible by training a deep model on controlled experimental data to predict viability at high accuracy, exploring what the model has learned, and applying it to an independent real-world dataset of an infectious pathogen. While the generalizability of this computational framework needs to be assessed in much more detail, we demonstrate that freely available data might be usable for relevant viability inferences in environmental, veterinary, and clinical settings.